The Interactive Fly

Zygotically transcribed genes

DNA damage checkpoint, meiotic crossover, nondisjunction and repair

Bloom syndrome helicase
RecQ helicase - repairs replication fork damage and double-strand breaks in mitosis - promotes repair through non-crossover
mechanisms - dissolution of double Holliday junctions - promotes meiotic crossover patterning and homolog disjunction

protein serine/threonine kinase - mitotic checkpoint control protein - inhibits ubiquitin ligase activity
of anaphase promoting complex (APC) preventing mitosis until all chromosomes are correctly attached to the mitotic spindle

mitotic checkpoint protein that serves as an essential protein required during normal mitotic progression to prevent premature sister
chromatid separation, missegreation and aneuploidy

crossover suppressor on 3 of Gowen
transverse element of the synaptonemal complex, a meiotic structure that regulates the repair of double-strand breaks (DSBs) into
crossovers or gene conversions - required for centromere separation in Pp1-87B RNAi oocytes
Chk1 homolog that functions in a developmentally regulated DNA replication/damage checkpoint operating
during the late syncytial divisions

loki (Chk2)
Chk2 homolog that functions in both the G1 and G2 checkpoint responses

meiotic 41
ATM/ATR kinase, phosphatidylinositol 3-kinase - essential for the DNA damage checkpoint in larval imaginal discs and neuroblasts -
required to delay mitosis in response to incomplete DNA replication in early nuclear divisions -
monitors double-strand-break repair during meiotic crossing over

encodes a multifunctional protein that plays critical roles in the response to DNA damage and telomere maintenance -
part of the MRN complex that which includes Mre11 and Rad50 - MRN senses DNA strand breaks and amplifies the signal and then conveys it to
downstream effectors, such as ATM (Telomere fusion in Drosophila) and p53, that regulate cell cycle checkpoints and DNA repair

DNA helicase involved in DNA repair - required for meiosis - the Drosophila homolog of the yeast DNA-repair protein Rad54

Transcription factor regulating cell cycle, DNA repair and apoptosis

Rad51-like (common alternative name: spindle A)
checkpoint protein essential for recombinational repair of double-stranded DNA breaks (DSBs) in somatic cells
and during meiosis in germ cells

telomere fusion (common alternative name: ATM)
a large, multifunctional protein kinase that regulates responses required for surviving DNA damage,
including functions such as DNA repair, apoptosis, and cell cycle checkpoints -
required for maintenance of normal telomeres and chromosome stability

bZip-domain transcription factor - transcriptionally upregulated by an autoregulatory loop - triggers apoptosis in competitively looser cells - regulates
translation and growth, delays development and is responsible for gene expression changes in mutant ribosomal proteins - induced by p53
following X-irradiation - partner of Inverted Repeat Binding Protein 18 - critical for repair of DNA breaks following transposase cleavage of DNA

The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila

The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. This study shows that the conserved kinase NHK-1 (Drosophila Ballchen) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, unrepaired DNA breaks formed during recombination were found to suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore it is proposed that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired (Lancaster, 2010).

Based on the evidence, a model is proposed in which DSBs formed during recombination suppress the activity of the conserved kinase NHK-1 through the meiotic recombination checkpoint to delay oocyte nuclear reorganisation from a recombination to a post-recombination phase. Although the evidence is mostly genetic or cytological, all data are so far consistent with this model. Nevertheless, the model is likely to be too simplistic and to represent only a part of the whole picture. For example, the possibility was not excluded that other checkpoint effectors are also involved in delaying meiotic progression. It is hopeed that the proposed model will prompt further investigation to fully uncover how the meiotic checkpoint is linked to meiotic progression (Lancaster, 2010).

How does NHK-1 kinase control this critical transition in meiosis? A previous study showed that NHK-1 directly controls karyosome formation through phosphorylation of BAF, a linker between the nuclear envelope and chromatin (Lancaster, 2007). Phosphorylation of BAF by NHK-1 releases meiotic chromosomes from tethering at the nuclear envelope to allow karyosome formation. Expression of non-phosphorylatable BAF disrupts karyosome formation, but not synaptonemal complex disassembly or condensin loading. Therefore, NHK-1 appears to control two independent pathways during nuclear reorganisation. This is consistent with a recent study showing that condensin is required for synaptonemal complex disassembly but not for karyosome formation. Karyosome formation and condensin loading are therefore likely to be two primary targets of NHK-1 activity (Lancaster, 2010).

Finally, this study in Drosophila is likely to have significant implications for understanding of meiotic regulation at a molecular level in other organisms, since the processes analyzed in this study are conserved among eukaryotes. The meiotic checkpoint that coordinates recombination events with meiotic progression is universally found across eukaryotes. Furthermore, NHK-1 is well conserved among animals, and karyosome-like clustering of meiotic chromosomes, as well as synaptonemal complex disassembly and condensin loading, is widely found in oocytes of various species including humans. In addition, this study has also suggested an involvement of NHK-1 in the DNA damage response during the mitotic cell cycle (Lancaster, 2010).

DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation

This study used germline stem cells (GSCs) in the Drosophila ovary to show that DNA damage retards stem cell self-renewal and lineage differentiation in a CHK2 kinase-dependent manner. Both heatshock-inducible endonuclease I-CreI expression and X-ray irradiation can efficiently introduce double-strand breaks in GSCs and their progeny, resulting in a rapid GSC loss and an accumulation of ill-differentiated GSC progeny. Elimination of CHK2 or its kinase activity can almost fully rescue the GSC loss and the progeny differentiation defect caused by DNA damage induced by I-CreI or X-ray. Surprisingly, checkpoint kinases ATM and ATR have distinct functions from CHK2 in GSCs in response to DNA damage. The reduction in BMP signaling and E-cadherin only makes limited contribution to DNA damage-induced GSC loss. Finally, DNA damage also decreases the expression of the master differentiation factor Bam in a CHK2-dependent manner, which helps explain the GSC progeny differentiation defect. Therefore, this study demonstrates, for the first time in vivo, that CHK2 kinase activation is required for the DNA damage-mediated disruption of adult stem cell self-renewal and lineage differentiation, and might also offer novel insight into how DNA damage causes tissue aging and cancer formation. It also demonstrates that inducible I-CreI is a convenient genetic system for studying DNA damage responses in stem cells (Ma, 2016).

Stem cells in adult tissues are responsible for generating new cells to combat against aging, and could also be cellular targets for tumor formation. Although aged stem cells have been shown to accumulate DNA damage, it remains largely unclear how DNA damage affects stem cell self-renewal and differentiation. A previous study has reported that upon weak irradiation apoptotic differentiated GSC progeny can prevent GSC loss by activating Tie-2 receptor tyrosine kinase signaling (Xing, 2015). This study shows that temporally introduced DNA double-stranded breaks cause premature GSC loss and slow down GSC progeny differentiation. Mechanistically, DNA damage causes GSC loss at least via two independent mechanisms, down-regulation of BMP signaling and E-cadherin-mediated GSC-niche adhesion as well as CHK2 activation- dependent GSC loss. In addition, CHK2 activation also decreases Bam protein expression by affecting its gene transcription and translation, slowing down CB differentiation into mitotic cysts and thus causing the accumulation of CB-like cells. Surprisingly, unlike in many somatic cell types, ATM, ATR, CHK1 and p53 do not work with CHK2 in DNA damage checkpoint control in Drosophila ovarian GSCs. Therefore, this study demonstrates that DNA damage-induced CHK2 activation causes premature GSC loss and also retards GSC progeny differentiation. The findings could also offer insight into how DNA damage affects stem cell-based tissue regeneration. In addition, this study also shows that the inducible I-CreI system is a convenient method for studying stem cell responses to transient DNA damage because it does not require any expensive irradiation equipment as the X-ray radiation does (Ma, 2016).

DNA damage normally leads to cell apoptosis to eliminate potential cancer- forming cells. This study, shows that transient DNA damage causes GSC loss not through apoptosis based on twopieces of experimental evidence: first, DNA-damaged GSCs are not positive for the cleaved Caspase-3, a widely used apoptosis marker; Second, forced expression of a known apoptosis inhibitor p35 does not show any rescue effect on DNA damage-induced GSC loss. Thus, DNA damage-induced GSC loss is likely due to self-renewal defects though the possibility could not be ruled out that other forms of cell death are responsible. p53 is known to be required for DNA damage-induced apoptosis from flies to humans. This study, however, demonstrates that p53 prevents the DNA damage-induced GSC loss. Vacating DNA-damaged GSCs from the niche via differentiation might allow their timely replacement and restoration of normal stem cell function. Therefore, the findings argue strongly that DNA damage primarily compromises self-renewal, thus causing GSC loss. Both niche-activated BMP signaling and E-cadherin-mediated cell adhesion are essential for GSC self-renewal. Consistent with the idea that DNA damage compromises GSC self-renewal, it significantly decreases BMP signaling activity and apical accumulation of E-cadherin in GSCs. Since constitutively active BMP signaling alone or in combination with E-cadherin overexpression can only moderately rescue GSC loss caused by DNA damage, it is concluded that decreased BMP signaling and apical E-cadherin accumulation might partly contribute to the DNA damage-induced GSC loss. Therefore, the findings suggest that DNA damage-mediated down-regulation of BMP signaling and E-cadherin-mediated adhesion only moderately contributes to the GSC loss (Ma, 2016).

DNA damage leads to checkpoint activation and cell cycle slowdown, thus giving more time for repairing DNA damage. In various cell types, ATM-CHK2 and ATR-CHK1 kinase pathways are responsible for DNA damage-induced checkpoint activation. During Drosophila meiosis, ATR, but not ATM, is required for checkpoint activity, indicating that ATM and ATR could have different functions in germ cells. Both ATR and CHK2 have been shown to be required for DNA damage-evoked checkpoint control in Drosophilagerm cells and embryonic cells, while CHK1 can control the entry into the anaphase of cell cycle in response to DNA damage, the G2-M checkpoint activation as well as the Drosophila midblastula transition (Ma, 2016).

This study has shown that these four checkpoint kinases function differently in GSCs. First, CHK2 is required for DNA damage-induced GSC loss, but is dispensable for normal GSC maintenance. Particularly, inactivation of its kinase activity can almost fully rescue DNA damage-induced GSC loss. Interestingly, inactivation of CHK2 function can also rescue the female germ cell defect caused by DNA damage in the mouse ovary, indicating that CHK2 function in DNA damage checkpoint activation is conserved at least in female germ cells. However, it remains unclear if CHK2 behaves similarly in mammalian stem cells in response to DNA damage. Second, ATM promotes GSC maintenance in the absence and presence of DNA damage. This is consistent with the finding that ATM is required for the maintenance of mouse male germline stem cells and hematopoietic stem cells. It will be interesting to investigate if ATM also prevents the oxidative stress in Drosophila GSCs as in mouse hematopoietic stem cells. Third, ATR is dispensable for normal GSC maintenance, but it protects GSCs in the presence of DNA damage. Although CHK2 and ATR behave similarly in DNA damage checkpoint control during meiosis and late germ cell development, they behave in an opposite way in GSCs in response to DNA damage. Finally, CHK1 is dispensable for GSC self-renewal in the absence and presence of DNA damage. Consistent with the current findings, the females homozygous for grp, encoding CHK1 in Drosophila, can still normally lay eggs, but those eggs could not develop normally. It will be of great interest in the future to figure out how CHK2 inactivation prevents DNA damage-induced GSC loss and how ATM and ATR inactivation promotes DNA damage-induced GSC loss at the molecular level. A further understanding of the functions of CHK2, ATM and ATR in stem cell response to DNA damage will help preserve aged stem cells and prevent their transformation into CSCs. DNA damage-evoked CHK2 activation retards GSC progeny differentiation by decreasing Bam expression at least at two levels This study has also revealed a novel mechanism of how DNA damage affects stem cell differentiation. Bam is a master differentiation regulator controlling GSC- CB and CB-cyst switches in the Drosophila ovary: CB-like single germ cells accumulate in bam mutant ovaries, whereas forced Bam expression sufficiently drives GSC differentiation. This study shows that DNA damage causes the accumulation of CB-like cells in a CHK2- dependent manner because CHK2 inactivation can fully rescue the germ cell differentiation defect caused by DNA damage. In addition, a heterozygous bam mutation can drastically enhance, and forced bam expression can completely repress, the DNA damage-induced germ cell differentiation defect, indicating that DNA damage disrupts Bam-dependent differentiation pathways. Consistently, Bam protein expression is significantly decreased in DNA damaged mitotic cysts in comparison with control ones. Interestingly, CHK2 inactivation can also fully restore Bam protein expression levels in the DNA-damaged mitotic cysts. Taken together, CHK2 activation is largely responsible forBam down-regulation in DNA damaged mitotic cysts, which can mechanistically explain the DNA damage-induced germ cell differentiation defect. It was further shown that DNA damage decreases Bam protein expression at least at two different levels. First, the bam transcription reporter bam-gfp was used to show that DNA damage decreases bamtranscription in CBs and mitotic cysts. Second, the posttranscriptional reporter Pnos-GFP-bam 3'UTR was generated to show that DNA damage decreases Bam protein expression via its 3'UTR in CBs and mitotic cysts at the level of translation. Although the detailed molecular mechanisms underlying regulation of Bam protein expression by DNA damage await future investigation, these findings demonstrate that DNA damage causes the GSC progeny differentiation defect by decreasing Bam protein expression at transcriptional and translational levels (Ma, 2016).

Taken together, these findings from Drosophila ovarian GSCs could offer important insight into how DNA damage affects stem cell-based tissue regeneration, and have also established Drosophila ovarian GSCs as a new paradigm for studying how DNA damage affects stem cell behavior at the molecular level. Because many stem cell regulatory strategies are conserved from Drosophila to mammals, what has been learned from this study should help understand how mammalian adult stem cells respond to DNA damage (Ma, 2016).

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that is termed "Inverted Repeat Binding Protein (IRBP) 18" and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability (Francis, 2016).

Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction

In most sexually reproducing organisms, crossover formation between homologous chromosomes is necessary for proper chromosome disjunction during meiosis I. During meiotic recombination, a subset of programmed DNA double-strand breaks (DSBs) are repaired as crossovers, with the remainder becoming noncrossovers. Whether a repair intermediate is designated to become a crossover is a highly regulated decision that integrates several crossover patterning processes, both along chromosome arms (interference and the centromere effect) and between chromosomes (crossover assurance). Because the mechanisms that generate crossover patterning have remained elusive for over a century, it has been difficult to assess the relationship between crossover patterning and meiotic chromosome behavior. This study showed that meiotic crossover patterning is lost in Drosophila melanogaster mutants that lack the Bloom syndrome helicase. In the absence of interference and the centromere effect, crossovers are distributed more uniformly along chromosomes. Crossovers even occur on the small chromosome 4, which normally never has meiotic crossovers. Regulated distribution of crossovers between chromosome pairs is also lost, resulting in an elevated frequency of homologs that do not receive a crossover, which in turn leads to elevated nondisjunction (Hatkevich, 2016).

Crossover interference, discovered by Sturtevant more than 100 years ago, is a meiotic crossover patterning phenomenon in which the presence of a crossover in one interval reduces the probability of a crossover in an adjacent interval. Studies in budding yeast, Arabidopsis, and mice revealed a subset of meiotic crossovers that do not show interference. These 'class II crossovers' are generated through a different pathway than most (class I) meiotic crossovers. In budding yeast, single-locus hotspot assays show that meiotic crossovers generated in the absence of the Bloom syndrome helicase (Blm) ortholog Sgs1 are formed primarily or exclusively by the class II pathway. This conclusion was partially derived from the observation that crossovers formed in sgs1 meiotic null mutants are not dependent upon Mlh1, a component of the meiosis-specific, class I Holliday junction resolvase. This study asked whether Drosophila Blm is also required to populate the class I pathway by determining whether crossovers generated in the absence of Blm are dependent upon MEI-9, the catalytic subunit of the presumptive Drosophila meiotic resolvase. Crossovers were measured in five adjacent intervals spanning most of 2L and part of 2R (for simplicity, referred to as 2L), a region comprising ~20% of the euchromatic genome. Loss of MEI-9 resulted in a >90% reduction in crossovers compared to wild-type flies. While Blm single mutants exhibit an ~30% decrease in crossovers on 2L, there is no additional reduction of crossovers in mei-9; Blm double mutants. Therefore, the crossovers that occur in Blm mutants do not require MEI-9, suggesting that they are generated through the class II pathway (Hatkevich, 2017).

The original distinction between crossovers generated by the class I and class II pathways is that only the former exhibit crossover interference. This study measured crossovers in three adjacent intervals on the X chromosome and calculated Stevens' measure of interference (I = 1 - [observed double crossovers/expected double crossovers]) between pairs of intervals. Interference was strong in wild-type flies (I = 0.89 and 0.85 for the two pairs of adjacent intervals) but was significantly reduced in Blm mutants (I = 0.19 and 0.29). Thus, without Blm helicase, crossovers are not dependent on the class I resolvase MEI-9, and interference among these crossovers is severely reduced or absent. This demonstrates that, as in S. cerevisiae, Drosophila Blm is required for the generation of crossovers through the class I pathway (Hatkevich, 2017).

Given the loss of interference, it was asked whether another important process that patterns crossovers along chromosomes arms-the centromere effect-is also lost in Blm mutants. This phenomenon, first reported by Beadle in 1932, is the suppression of crossover formation in centromere-proximal euchromatin. To quantify the centromere effect, a measure, CE, was devised that is analogous to I as a measure of interference in that CE = 1 - (observed/expected), where observed is the number of crossovers counted in the interval and expected is the number expected in a random distribution. In wild-type females, the interval between pr and cn, which spans the chromosome 2 centromere, had a CE of 0.89, consistent with a strong centromere effect. In Blm mutants this was reduced to 0.36 (p < 0.0001). The centromere effect was much weaker on the X chromosome due to the larger block of heterochromatin that moves the euchromatin further from the centromere. CE in a centromere-spanning interval on the X was 0.29 in wild-type flies, but it was reduced to -0.04 in Blm mutants (Hatkevich, 2017).

Loss of interference and the centromere effect in Blm mutants allows assessment of the consequences of loss of crossover patterning along chromosome arms. Because these crossover patterning processes are responsible for the overall crossover distribution along each chromosome arm, the effect of these losses on crossover distribution was assessed along entire arms. In wild-type flies, genetic length was not proportional to physical length, with crossover density being higher in the middle of each arm. On both the X and 2L, crossover distribution in Blm mutants was significantly different from the wild-type distribution. Instead, crossovers in Blm mutants appeared to be distributed in a manner more proportional to physical length. In wild-type flies, nine of the ten intervals were examined had significantly different numbers of crossovers than expected if genetic distance is proportional to physical distance, but in Blm mutants only three intervals were significantly different than this expectation. The deviations in three intervals in Blm mutants may reflect residual crossover patterning; however, the 2L crossover distributions in mei-9; Blm double mutants and in mutants carrying the helicase-dead allele BlmE866K were even more closely proportional to physical length, suggesting that the departures from proportionality in Blm-null mutants may be an effect of strain background (Hatkevich, 2017).

A particularly extreme case of crossover patterning was examined: the absence of crossovers on the small chromosome 4 of Drosophila melanogaster. There are never crossovers on this chromosome in wild-type females, but there have been reports of conditions that do result in crossovers. One study found crossovers in 4-derived sequences when they were translocated to chromosome 3. This result suggests that the absence of crossovers on 4 may be a consequence of crossover patterning processes. Support for this idea came from whole-genome sequencing that revealed the presence of noncrossover gene conversion on 4, indicating that double-strand breaks (DSBs) are made on 4 and, therefore, it is the repair process that is regulated to prevent crossovers (Hatkevich, 2017).

Recombination was scored between two markers near opposite ends of the genome sequence assembly of 4. As expected, no crossovers between these markers were recovered in wild-type females; however, in Blm mutants, ten crossovers were recovered among 3,106 progeny. Blm mutants have spontaneous mitotic crossovers in the male germline. To ensure that the crossovers that were observed were meiotic, meiotic DSBs were eliminated; no crossovers were observed in this case. It is concluded that the absence of crossovers on chromosome 4 in wild-type females is a result of active meiotic crossover patterning processes that are intertwined with the class I crossover pathway. This is most likely due to the centromere effect, consistent with the observation that crossovers occur in 4 sequences that are translocated to a genomic region further from the centromere. Interference should not be applicable to 4 because there are no initial crossover designations to discourage nearby additional designations (Hatkevich, 2017).

Although crossovers in Blm mutants were distributed approximately evenly along X and 2L, and also occurred on chromosome 4, average crossover density was not the same between these chromosomes. In both wild-type females and Blm mutants, crossover density was higher on the X than on 2L, and it was lower still on chromosome 4 in Blm mutants. Possible explanations for this include different DSB densities, different strengths of crossover patterning (e.g., the weaker centromere effect on the X compared to chromosome 2), and residual crossover patterning in Blm mutants (Hatkevich, 2017).

The results above show that crossover patterning along chromosomes is lost or severely reduced in Blm mutants. Crossover patterning also occurs between chromosomes. It has been reported that, in a grasshopper species with a large range of chromosome sizes, every pair of homologous chromosomes always had at least one chiasma (the cytological manifestation of a crossover), called the obligate chiasma. The occurrence of an obligate chiasma suggests that there is an active process, referred to as crossover assurance, that monitors the designation of crossovers on each chromosome. To determine whether loss of Blm affects crossover assurance, the observed and expected frequencies of E0 tetrads (homologous chromosome pairs with no crossovers) were compared. In wild-type flies, the observed E0 frequency for the X chromosome (0.112) was less than half the frequency expected based on Poisson distribution (0.285, p < 0.0001), indicating that crossover assurance is present, but it is not absolute. Crossover assurance is significantly reduced or absent in Blm mutants (p < 0.0001 compared to wild-type), resulting in the observed E0 frequency (0.514) being similar to the expected frequency (0.550) (Hatkevich, 2017).

The results described above reveal that the three major aspects of crossover patterning that occur along and among chromosomes-interference, the centromere effect, and assurance-are significantly decreased or eliminated when Blm helicase is absent. This suggests an inability to make or execute the crossover/noncrossover decision. Mapping of noncrossover gene conversion events in wild-type flies through whole-genome sequencing reveals a flat distribution along each of the major chromosome arms, similar to the distribution of crossovers in Blm mutants. Noncrossovers do not participate in interference and are not subject to the centromere effect. These findings suggest that DSBs are evenly distributed along each arm, at least at the megabase scales at which crossovers were mapped. In wild-type flies, crossover patterning processes act on this distribution such that events in the central regions of the major chromosome arms have a higher probability of being designated to become crossovers, and those on chromosome 4 are never so designated. Regulated crossover designation is lost in Blm mutants, and, as a result, every DSB repair event has the same probability of becoming a crossover, regardless of where along the chromosome it is located (Hatkevich, 2017).

The results argue that meiotic DSB repair in Blm mutants occurs outside of the predominant meiotic recombination pathway and that this results in the loss of regulated crossover designation and patterning. What are the consequences of these losses on meiosis? In Blm mutants, nondisjunction of the X chromosome is elevated 30-fold. In wild-type females, most X chromosomes that nondisjoin did not have any crossovers, had only a single crossover that was distal, or had a centromere-proximal crossover. X chromosomes were examined that nondisjoined in Blm mutants. In 33 of 34 cases, the nondisjoined chromosomes had no crossovers; the remaining case had a single crossover in the most centromere-proximal interval (Hatkevich, 2017).

Most X nondisjunction in Blm mutants occurs between chromosomes that did not experience a crossover. The incidence of E0 X chromosomes was elevated in Blm mutants due to a combination of decreased crossover frequency and loss of assurance. To separate these effects, Blm rec double mutants were examined. REC, the Drosophila ortholog of MCM8, is required in the class I crossover pathway. Crossovers are greatly reduced in rec single mutants but were elevated above wild-type levels in Blm rec double mutants. The reasons for this elevation are unknown, but they may be related to the poorly understood role of REC in the noncrossover pathway. Despite the elevated crossover frequency, nondisjunction rates were similar in Blm mutants and Blm rec double mutants. Like Blm single mutants, Blm rec double mutants exhibited a loss of interference, the centromere effect, and crossover assurance, and crossovers occurred on chromosome 4. These results argue that the elevated nondisjunction seen in Blm mutants is due primarily to the loss of crossover patterning (Hatkevich, 2017).

Interference, the centromere effect, and the obligate chiasma were all described more than 80 years ago, but the mechanisms behind these phenomena remain unknown. These phenomena are entwined in the class I crossover pathway, but it is unclear whether they are generated independently within this pathway or are merely different manifestations of a single regulatory process. Mathematical modeling has suggested that an obligatory crossover is ensured by a combination of interference and other features of the class I pathway, so these processes may be inter-dependent. The centromere effect may be an augmentation that reinforces interference by pushing crossovers toward the middle of the arm. However, since the telomere effect in Drosophila was far weaker than the centromere effect, it seems likely that the centromere effect is an independent phenomenon that functions to prevent proximal crossovers, presumably because these can induce nondisjunction. Only a single case was identified of a proximal crossover in the set of nondisjoined chromosomes that were analyzed, but this was a significant increase from the frequency in wild-type females (one case in ~2,900 progeny in Blm mutants compared to six cases from ~600,000 progeny of wild-type females (Hatkevich, 2017).

The meiotic function of Drosophila Blm appears to be similar to the role of S. cerevisiae Sgs1 in allowing recombination intermediates to populate the class I crossover pathway, but this is not conserved in some other species. In Arabidopsis, redundant Blm paralogs prevent class II crossovers, perhaps by promoting noncrossover repair, but are not required for class I crossovers. The C. elegans ortholog, HIM-6, does have a role in making class I crossovers; however, this occurs after normal crossover designation. This is not unlike Drosophila mei-9 mutants, where crossover designation is intact but crossover formation is impaired, resulting in the few crossovers that are made having a wild-type distribution (Hatkevich, 2017).

In summary, this study has assessed the importance of crossover patterning in meiosis by exploiting the loss of patterning in Drosophila mutants lacking Blm helicase. In wild-type females, the primary meiotic recombination pathway incorporates the centromere effect and interference to promote patterned designation of which events will become crossovers. Strong crossover assurance means that most homologous chromosomes have a crossover that ensures their disjunction, but the few achiasmate pairs are still segregated accurately by the achiasmate segregation system. Blm is essential for entrance into this meiosis-specific class I repair pathway; in Blm mutants, repair instead occurs through the class II pathway. These crossovers lead to chiasmata that are competent to promote accurate disjunction. However, because crossover patterning is lost, there is an elevated frequency of chromosomes at risk for nondisjunction (primarily achiasmate chromosomes, but possibly also chromosomes with very proximal crossovers) (Hatkevich, 2017).

Annealing of complementary DNA sequences during double-strand break repair in Drosophila is mediated by the ortholog of SMARCAL1

DNA double-strand breaks (DSBs) pose a serious threat to genomic integrity. If unrepaired, they can lead to chromosome fragmentation and cell death. If repaired incorrectly, they can cause mutations and chromosome rearrangements. DSBs are repaired using end-joining or homology-directed repair strategies, with the predominant form of homology-directed repair being synthesis-dependent strand annealing (SDSA). SDSA is the first defense against genomic rearrangements and information loss during DSB repair, making it a vital component of cell health and an attractive target for chemotherapeutic development. SDSA has also been proposed to be the primary mechanism for integration of large insertions during genome editing with CRISPR/Cas9. Despite the central role for SDSA in genome stability, little is known about the defining step: annealing. It was hypothesized that annealing during SDSA is performed by the annealing helicase SMARCAL1, which can anneal RPA-coated single DNA strands during replication-associated DNA damage repair. The study utilized genetic tools in Drosophila melanogaster to test whether the fly ortholog of SMARCAL1, Marcal1, mediates annealing during SDSA. Repair that requires annealing is significantly reduced in Marcal1 null mutants in both synthesis-dependent and synthesis-independent (single-strand annealing) assays. Elimination of the ATP binding activity of Marcal1 also reduces annealing-dependent repair, suggesting that the annealing activity requires translocation along DNA. Unlike the null mutant, however, the ATP binding-defect mutant shows reduced end-joining, shedding light on the interaction between SDSA and end-joining pathways. (Holsclaw, 2017).

Conserved Function Of Drosophila Fancd2 Monoubiquitination In Response To Double-Strand DNA Breaks
Fanconi Anemia (FA) genes play key roles in metazoan DNA damage responses, and human FA mutations cause numerous disease phenotypes. In human cells, activating monoubiquitination of the FA protein Fancd2 occurs following diverse DNA damage stimuli. Monoubiquitinated Fancd2 forms nuclear foci to recruit additional repair factors. Fancd2 animal models to date have focused on molecular nulls or whole gene knockdown, leaving the specific in vivo role of monoubiquitination unclear. Using a point mutant in a conserved residue, Drosophila Fancd2 monoubiquitination has been linked to a mitosis-specific DNA double-strand break (DSB) response. In this context, CRISPR/Cas9 was used to generate the first animal model of an endogenous mutation in the conserved monoubiquitination site (fancd2K595R). This study expands on the characterization of fancd2K595R. Additional Drosophila tools were introduced and characterized to study fancd2, including new mutant alleles and GFP-tagged rescue transgenes. Using these new reagents, the impact was shown of Drosophila Fancd2 on organismal and cell viability, as well as on repair protein localization, in the presence or absence of DSBs. These findings expand understanding of FA gene function in vivo and provide useful reagents for DNA repair research (Clay, 2022).

Genetic variation in P-element dysgenic sterility is associated with double-strand break repair and alternative splicing of TE transcripts

The germline mobilization of transposable elements (TEs) by small RNA mediated silencing pathways is conserved across eukaryotes and critical for ensuring the integrity of gamete genomes. However, genomes are recurrently invaded by novel TEs through horizontal transfer. These invading TEs are not targeted by host small RNAs, and their unregulated activity can cause DNA damage in germline cells and ultimately lead to sterility. This study used hybrid dysgenesis-a sterility syndrome of Drosophila caused by transposition of invading P-element DNA transposons-to uncover host genetic variants that modulate dysgenic sterility. Using a panel of highly recombinant inbred lines of Drosophila melanogaster, two linked quantitative trait loci (QTL) were identified that determine the severity of dysgenic sterility in young and old females, respectively. Ovaries of fertile genotypes exhibit increased expression of splicing factors that suppress the production of transposase encoding transcripts, which likely reduces the transposition rate and associated DNA damage. It was also shown that fertile alleles are associated with decreased sensitivity to double-stranded breaks and enhanced DNA repair, explaining their ability to withstand high germline transposition rates. Together, this work reveals a diversity of mechanisms whereby host genotype modulates the cost of an invading TE, and points to genetic variants that were likely beneficial during the P-element invasion (Lama, 2022).

Dicentric chromosome breakage in Drosophila melanogaster is influenced by pericentric heterochromatin and occurs in non-conserved hotspots

Chromosome breakage plays an important role in the evolution of karyotypes, and can produce deleterious effects within a single individual, such as aneuploidy or cancer. Forces that influence how and where chromosomes break are not fully understood. In humans, breakage tends to occur in conserved hotspots called common fragile sites (CFS), especially during replication stress. By following the fate of dicentric chromosomes in Drosophila melanogaster this study found that breakage under tension also tends to occur in specific hotspots. The experimental approach was to induce sister chromatid exchange in a ring chromosome to generate a dicentric chromosome with a double chromatid bridge. In the following cell division, the dicentric bridges may break. The breakage patterns were analyzed of three different ring-X chromosomes. These chromosomes differ by the amount and quality of heterochromatin they carry as well as their genealogical history. For all three chromosomes, breakage occurs preferentially in several hotspots. Surprisingly, it was found that the hotspot locations are not conserved between the three chromosomes: each displays a unique array of breakage hotspots. The lack of hotspot conservation, along with a lack of response to aphidicolin, suggests that these breakage sites are not entirely analogous to CFS and may reveal new mechanisms of chromosome fragility. Additionally, the frequency of dicentric breakage and the durability of each chromosome's spindle attachment varies significantly between the three chromosomes and is correlated with the origin of the centromere and the amount of pericentric heterochromatin. It is suggested that different centromere strengths could account for this (Hill, 2023).

DNA polymerase theta suppresses mitotic crossing over

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol theta) to use resected, 3' single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. This study shows that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol theta in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol theta and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol theta or SLX4 to survive. The current results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. Mitotic crossovers were measured in flies after a Cas9-induced chromosome break; this mutagenic form of repair is increased in the absence of Pol theta. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. This work argues that Pol theta can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells (Carvajal-Garcia, 2021).

Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo

The DNA damage checkpoint is crucial to protect genome integrity. However, the early embryos of many metazoans sacrifice this safeguard to allow for rapid cleavage divisions that are required for speedy development. At the mid-blastula transition (MBT), embryos switch from rapid cleavage divisions to slower, patterned divisions with the addition of gap phases and acquisition of DNA damage checkpoints. The timing of the MBT is dependent on the nuclear-to-cytoplasmic (N/C ratio) and the activation of the checkpoint kinase, Chk1. How Chk1 activity is coupled to the N/C ratio has remained poorly understood. This study shows that dynamic changes in histone H3 availability in response to the increasing N/C ratio control, Chk1 activity, and thus time the MBT in the Drosophila embryo. Excess H3 in the early cycles was shown to interfere with cell-cycle slowing independent of chromatin incorporation. The N-terminal tail of H3 acts as a competitive inhibitor of Chk1 in vitro and reduces Chk1 activity in vivo. Using a H3-tail mutant that has reduced Chk1 inhibitor activity, this study showed that the amount of available Chk1 sites in the H3 pool controls the dynamics of cell-cycle progression. Mathematical modeling quantitatively supports a mechanism where titration of H3 during early cleavage cycles regulates Chk1-dependent cell-cycle slowing. This study defines Chk1 regulation by H3 as a key mechanism that coordinates cell-cycle remodeling with developmental progression (Shindo, 2021).

The microtubule- and PP1-binding activities of Drosophila melanogaster Spc105 control the kinetics of SAC satisfaction

KNL1 is a large intrinsically disordered kinetochore (KT) protein that recruits spindle assembly checkpoint (SAC) components to mediate SAC signaling. The N-terminal region (NTR) of KNL1 possesses two activities that have been implicated in SAC silencing: microtubule (MT) binding and protein phosphatase 1 (PP1) recruitment. The NTR of D. melanogaster KNL1 (Spc105) has never been shown to bind MTs nor to recruit PP1. Furthermore, the phospho-regulatory mechanisms known to control SAC protein binding to KNL1 orthologues is absent in D. melanogaster. This study resolved these apparent discrepancies are resolved using in vitro and cell based-assays. A phospho-regulatory circuit, which utilizes Aurora B kinase (ABK), promotes SAC protein binding to the central disordered region of Spc105 while the NTR binds directly to MTs in vitro and recruits PP1-87B to KTs in vivo. Live-cell assays employing an optogenetic oligomerization tag, and deletion/chimera mutants are used to define the interplay of MT- and PP1-binding by Spc105 and the relative contributions of both activities to the kinetics of SAC satisfaction (Audett, 2021).

dRTEL1 is essential for the maintenance of Drosophila male germline stem cells

Stem cells have the potential to maintain undifferentiated state and differentiate into specialized cell types. Despite numerous progress has been achieved in understanding stem cell self-renewal and differentiation, many fundamental questions remain unanswered. In this study, dRTEL1, the Drosophila homolog of Regulator of Telomere Elongation Helicase 1 (CG4078), was identified as a novel regulator of male germline stem cells (GSCs). Genome-wide transcriptome analysis and ChIP-Seq results suggest that dRTEL1 affects a set of candidate genes required for GSC maintenance, likely independent of its role in DNA repair. Furthermore, dRTEL1 prevents DNA damage-induced checkpoint activation in GSCs. Finally, dRTEL1 functions to sustain Stat92E protein levels, the key player in GSC maintenance. Together, these findings reveal an intrinsic role of the DNA helicase dRTEL1 in maintaining male GSC and provide insight into the function of dRTEL1 (Yang, 2021).

The Role of Drosophila CtIP in Homology-Directed Repair of DNA Double-Strand Breaks

DNA double-strand breaks (DSBs) are a particularly genotoxic type of DNA damage that can result in chromosomal aberrations. Thus, proper repair of DSBs is essential to maintaining genome integrity. DSBs can be repaired by non-homologous end joining (NHEJ), where ends are processed before joining through ligation. Alternatively, DSBs can be repaired through homology-directed repair, either by homologous recombination (HR) or single-strand annealing (SSA). Both types of homology-directed repair are initiated by DNA end resection. In cultured human cells, the protein CtIP has been shown to play a role in DNA end resection through its interactions with CDK, BRCA1, DNA2, and the MRN complex. To elucidate the role of CtIP in a multicellular context, CRISPR/Cas9 genome editing was used to create a DmCtIPΔ allele in Drosophila melanogaster. Using the DSB repair reporter assay direct repeat of white (DR-white), a two-fold decrease in HR in DmCtIPΔ/Δ mutants was observed when compared to heterozygous controls. However, analysis of HR gene conversion tracts (GCTs) suggests DmCtIP plays a minimal role in determining GCT length. To assess the function of DmCtIP on both short (~550 bp) and long (~3.6 kb) end resection, modified homology-directed SSA repair assays were implemented, resulting in a two-fold decrease in SSA repair in both short and extensive end resection requirements in the DmCtIPΔ/Δ mutants compared to heterozygote controls. Through these analyses, the importance was affirmed of end resection on DSB repair pathway choice in multicellular systems, the function of DmCtIP in short and extensive DNA end resection was described, and the impact of end resection on GCT length during HR was determined (Yannuzzi, 2021).

Drosophila p53 isoforms have overlapping and distinct functions in germline genome integrity and oocyte quality control

p53 gene family members in humans and other organisms encode a large number of protein isoforms whose functions are largely undefined. Using Drosophila as a model, it was found that a p53B isoform is expressed predominantly in the germline where it colocalizes with p53A into subnuclear bodies. It is only p53A, however, that mediates the apoptotic response to ionizing radiation in the germline and soma. In contrast, p53A and p53B are both required for the normal repair of meiotic DNA breaks, an activity that is more crucial when meiotic recombination is defective. In oocytes with persistent DNA breaks p53A is also required to activate a meiotic pachytene checkpoint. These findings indicate that Drosophila p53 isoforms have DNA lesion and cell type-specific functions, with parallels to the functions of mammalian p53 family members in the genotoxic stress response and oocyte quality control (Chakravarti, 2022).

The Drosophila melanogaster genome has a single p53 family member. Similar to human p53 (TP53), it has a C terminal oligomerization domain (OD), a central DNA-binding domain (DBD) and an N terminal transcriptional activation domain (TAD), and functions as a tetrameric transcription factor. This single p53 gene expresses four mRNAs that encode three different protein isoforms. A 44 kD p53A protein isoform was the first to be identified and is the most well characterized. Later RNA-Seq and other approaches revealed that alternative promoter usage and RNA splicing results in a 56 kD p53B protein isoform, which differs from p53A by a 110 amino acid longer N-terminal TAD that is encoded by a unique p53B 5' exon. Because the p53A isoform differs from p53B by a shorter N terminus, p53A is also known as ΔNp53. A p53C transcript starts at a different promoter than p53A but is predicted to encode the same 44 kD protein. A short p53E mRNA isoform is predicted to encode a protein of 38 kD that contains the DNA-binding domain but lacks the longer N-terminal TADs of p53A and p53B (Chakravarti, 2022).

Like its human ortholog, Drosophila p53 regulates apoptosis in response to genotoxic stress and mediates other stress responses and developmental processes. To promote apoptosis, p53 induces transcription of several proapoptotic genes at one locus called H99. Early analyses of p53 function in apoptosis focused on the p53A isoform because the others had yet to be discovered. Using BAC rescue transgenes that were mutant for either p53A or p53B, previous work showed that in larval tissues it is the shorter p53A, and not p53B, that is both necessary and sufficient for the apoptotic response to DNA damage caused by ionizing radiation. In contrast, when each isoform was overexpressed, p53B was much more potent than p53A at inducing proapoptotic gene transcription and the programmed cell death response, likely because of the longer p53B TAD. Other evidence suggests that p53B may regulate tissue regeneration and has a redundant function with p53A to regulate autophagy in response to oxidative stress. It is largely unknown, however, why the Drosophila genome encodes a separate p53B isoform and what its array of functions are (Chakravarti, 2022).

The p53 gene family is ancient with orthologs found in the genomes of multiple eukaryotes, including single-celled Choanozoans, which are thought to be the ancestors of multicellular animals. Evidence suggests that the ancestral function of the p53 gene family was in the germline, with later evolution of tumor suppressor functions in the soma. In mammals, p63 mediates a meiotic pachytene checkpoint arrest in response to DNA damage or chromosome defects, and also induces apoptosis of a large number of oocytes with persistent defects, thereby enforcing an oocyte quality control. It has been shown that in the Drosophila germline p53 regulates stem cell divisions, responds to programmed meiotic DNA breaks, and represses mobile elements. This study has uncovered that the Drosophila p53A and p53B isoforms have overlapping and distinct functions during oogenesis to protect genome integrity and mediate the meiotic pachytene checkpoint arrest, with parallels to the germline function of mammalian p53 family members in oocyte quality control (Chakravarti, 2022).

This study found that the Drosophila p53B protein isoform is more highly expressed in the germline where it colocalizes with a shorter p53A isoform in subnuclear bodies. Despite this p53B germline expression, it is the p53A isoform that was necessary and sufficient for the apoptotic response to IR in both the germline and soma. Although apoptosis is repressed in meiotic oocytes and endocycling nurse cells, it was found that both p53 isoforms are required in these cells for the timely repair of meiotic DNA breaks. The role of the p53 isoforms in DNA repair was cell type specific, with p53B playing the most prominent role in the nurse cells, whereas both p53B and p53A were required in the oocyte. The data has also uncovered a requirement for the Drosophila p53A isoform in the meiotic pachytene checkpoint response to unrepaired DNA breaks. Overall, these data suggest that Drosophila p53 isoforms have evolved overlapping and distinct functions to mediate different responses to different types of DNA damage in different cell types. These findings are relevant to understanding the evolution of p53 isoforms, and have revealed interesting parallels to the function of mammalian p53 family members in oocyte quality control (Chakravarti, 2022).

p53 isoforms colocalized to subnuclear bodies in the Drosophila male and female germline. This finding is consistent with a previous study that reported p53 bodies in the Drosophila male germline, although that study did not examine individual isoforms. It is deemed likely that these p53 bodies form by phase separation, an hypothesis that remains to be formally tested. Drosophila p53 subnuclear bodies are reminiscent of human p53 protein localization to subnuclear PML bodies. Evidence suggests that trafficking of human p53 protein through PML bodies mediates p53 post-translational modification and function, although the relationship between nuclear trafficking and the functions of different p53 isoforms has not been fully evaluated. Similarly, a decline was observed in abundance of p53B within p53 bodies in germarium region 2a, followed by a restoration of p53B within bodies in region 3. This fluctuation of p53B in bodies temporally correlates with the onset of meiotic DNA breaks in region 2a and their repair in regions 2b - 3. These observations are consistent with the idea that nuclear trafficking of p53B out of bodies may mediate its response to meiotic breaks, although it is also possible that p53B is degraded and rapidly resynthesized during this 24 hr period. Future analysis of Drosophila p53 bodies will help to define how p53 isoform trafficking mediates the response to genotoxic and other stresses (Chakravarti, 2022).

TUNEL labeling indicated that p53A is necessary and sufficient for apoptosis in both the germline and soma. IR induced apoptosis to a similar frequency in p53+ (A+B+) wild type and p53B41.5 (A+B-) mutants, whereas the frequency of apoptosis in p53A2.3 (A-B+) mutants was equivalent to that of p535A-1-4 (A-B-) null and unirradiated controls. Consistent with this, hid-GFP reporter expression was not induced by IR in the p535A-1-4 (A-B-) null mutant, whereas IR-induced hid-GFP expression in the p53B41.5 (A+B-) mutant was equivalent to p53+ (A+B+) wild type, indicating that the p53A isoform is required for the transcriptional response to IR-induced DNA breaks. It is interesting to note that while germline cystocytes in germarium region one apoptosed after IR, their ancestor GSCs and descendent meiotic cells did not. The observed IR-induced expression of the hid-GFP promoter reporter in GSCs is consistent with previous evidence that apoptosis is repressed in these stem cells downstream of hid transcription by the miRNA bantam. How meiotic cells repress apoptosis is not known, although it is crucial that they do so because they have programmed DNA breaks. Together, these data suggest that p53A is necessary and sufficient for induction of proapoptotic gene expression and apoptosis in response to IR-induced DNA breaks in the soma and germline (Chakravarti, 2022).

While this manuscript was in preparation, it was reported that p53A and p53B both participate in the apoptotic response to IR in the ovary (Park, 2019). That study used the GAL4/ UAS system to express either p53A or p53B rescue transgenes in a p53 null background. In contrast, this study created and analyzed loss-of-function, isoform-specific alleles at the endogenous p53 locus, which is believed to more accurately reflect the physiological function of p53 isoforms. The conclusion, therefore, is favored that it is the p53A isoform that has the primary function of mediating the apoptotic response to IR in the soma and germline (Chakravarti, 2022).

In the absence of IR, there was a lower but detectable hid-GFP expression at the onset of meiosis in germarium region 2. This region 2 expression was dependent on p53 and formation of meiotic breaks by Mei-W68, consistent with previous reports that used a rpr-GFP reporter to show that p53 responds to meiotic DNA breaks. This low level of hid-GFP expression in region two without IR was similar between p53+ (A+B+) wild type and p53B41.5 (A+B-) mutants, suggesting that the p53A transcription factor activity responds to meiotic DNA breaks. The results for the p53A2.3 (A-B+) mutant were not informative, however, because in that mutant hid-GFP expression was constitutively higher than wild type beginning in early region 1 of the germarium. γ-H2Av labeling was not observed before late region 1/ region 2 a indicating that this low-level activity of p53B is not a response to DNA breaks. While further experiments are required to define the mechanism, a cogent hypothesis is that in the absence of the p53A subunit p53B homotetramers have somewhat higher basal activity. This hypothesis is consistent with previous evidence that the p53B isoform with a longer transactivation domain is a much stronger transcription factor than p53A, and that p53A and p53B can form heterocomplexes. It is also consistent with evidence that the shorter p53 isoforms in humans and other organisms repress the transcriptional activity of longer isoforms in heterotetramers. It is important to note, however, that while hid expression was higher in the p53A mutants than in wild type, it was not associated with apoptosis. Overall, while the hid-GFP reporter evidence suggests that p53A responds to meiotic DNA breaks, it is unclear whether this low-level activation of p53A transcription factor activity is related to its role in meiotic DNA break repair or checkpoint activation, which is discussed further below (Chakravarti, 2022).

The evidence suggests that both p53 isoforms are required for the timely repair of meiotic DNA breaks in the Drosophila female germline. p53 null and isoform-specific mutants had a persistent germline DNA break phenotype that was dependent on the creation of double-strand DNA breaks by Mei-W68. Further consistent with a role in meiotic DNA break repair, p53 mutants had an increased number of cells with γ-H2Av foci beginning in germarium stage 2a, the time when Mei-W68 induces programmed meiotic DNA breaks. Moreover, the number of persistent breaks per cell was higher in oocyte and adjacent nurse cell, the presumptive pro-oocyte, which are known to have more meiotic breaks. This p53 DNA break mutant phenotype is similar to that of okra (RAD54L) and other genes required for meiotic break repair and was enhanced in okra; p53 double mutants. It was previously shown using p53 null alleles that p53 also protects the germline genome by restraining mobile element activity, but this study did not evaluate whether one or both of the p53 isoforms are required for this function. Overall, the current data strongly suggest that both p53 isoforms have an important role in the repair of meiotic DNA breaks (Chakravarti, 2022).

This analysis also revealed that p53 isoforms have overlapping and distinct requirements for meiotic break repair in different cell types. Both p53A and p53B were required in the oocyte, whereas p53B played the more prominent role in nurse cells, even though nurse cells express both p53A and p53B isoforms. This differential requirement for p53 isoforms may reflect differences in how meiotic breaks are repaired in nurse cells versus oocytes. While it is not known whether DNA repair pathways differ between nurse cells and oocytes, evidence suggests that the creation of meiotic breaks does, with breaks in pro-oocytes but not pro-nurse cells depending on previous SC formation. Important questions motivated by the current results are how distinct responses to DNA damage in different cells are determined by different types of DNA lesions, checkpoint signaling and repair pathways, and p53 isoform structure (Chakravarti, 2022).

The consequences of p53 null and isoform-specific alleles for oogenesis were also similar to okra mutants in that they caused reduced female fertility and defects in eggshell patterning and synthesis. Previous evidence suggested that defective meiotic DNA break repair causes these maternal effect phenotypes in part through disrupting patterning signals from the oocyte to somatic follicle cells. The maternal effect on egg hatch rates, however, was much more severe in the okra mutants, which were completely female sterile, consistent with previous studies. Thus, although the p53 and okra null mutants had similar levels of germline DNA damage, the severity of their maternal-effect on egg patterning and embryo viability differ, suggesting that some of their pleiotropic effects on oogenesis are distinct. Together, the results indicate that defects in repair of meiotic DNA breaks in both p53 and okra mutant females negatively impact embryo patterning and female fertility (Chakravarti, 2022).

The requirement for Drosophila p53 in the repair of meiotic DNA breaks is consistent with evidence from other organisms that p53 has both indirect and direct roles in DNA repair. It is known that Drosophila p53 and specific isoforms of human p53 induce the expression of genes that are required for different types of DNA repair. p53 also acts locally at DNA breaks in a variety of organisms, including humans, where it can mediate the choice between HR versus non-homologous end joining (NHEJ) repair. In fact, it has been shown that human p53 directly associates with RAD54 at DNA breaks to regulate HR repair, consistent with the finding that p53; okra (RAD54L) double mutants have severe DNA repair defects. Moreover, the C. elegans p53 ortholog CED-4 localizes to DNA breaks to promote HR and inhibit NHEJ repair in the germline. Although the hid-GFP reporter indicated that meiotic DNA breaks induce a low level of p53A transcription factor activity, Hid has no known role in DNA repair, and it remains unknown whether p53-regulated expression of DNA repair genes is required for the timely repair of meiotic DNA breaks. It is deemed likely that the persistent DNA damage that was observed in the germline of Drosophila p53 mutants may, in part, reflect a local requirement for p53 protein isoforms to regulate meiotic DNA repair. Important remaining questions include whether different p53 isoforms participate indirectly in DNA repair by inducing transcription and directly at DNA breaks to influence the choice among different DNA repair pathways (Chakravarti, 2022).

This study has also uncovered a requirement for Drosophila p53 in the meiotic pachytene checkpoint. This function was isoform-specific, with p53A, but not p53B, being required for full checkpoint activation in oocytes with persistent DNA breaks. The failure to engage the pachytene checkpoint in the majority of okra; p53A2.3 double mutant oocytes is more striking given that these cells had more severe DNA repair defects than the okra single mutants that strongly engaged the checkpoint. While the pachytene arrest was compromised to similar extents in okra; p53 null and okra; p53A2.3 mutants, some egg chambers in both genotypes did engage a pachytene arrest. This observation suggests that there are p53-independent mechanisms that also activate the checkpoint, perhaps in response to secondary defects in chromosome structure, which are known to independently trigger the pachytene checkpoint in flies and mammals. Moreover, although the pachytene checkpoint was strongly compromised in the p53 null and p53A mutant alleles, it did not suppress okra female sterility, suggesting that other mechanisms ensure that oocytes with excess DNA damage do not contribute to future generations. Altogether, the results indicate that p53A is required for both DNA repair and full pachytene checkpoint activation in the oocytes (Chakravarti, 2022).

Evidence suggests that the ancient function of the p53 family was of a p63-like protein in the germline. Consistent with this, the findings in Drosophila have parallels to mammals where the TAp63α isoform and p53 mediate a meiotic pachytene checkpoint arrest, and the apoptosis of millions of oocytes that have persistent defects. The current evidence suggests that the different isoforms of the sole p53 gene in Drosophila may subsume the functions of vertebrate p53 and p63 paralogs to protect genome integrity and mediate the pachytene arrest. Unlike p53 and p63 in mammals, however, Drosophila p53 does not trigger apoptosis of defective oocytes. Instead, the activation of the pachytene checkpoint disrupts egg patterning, resulting in inviable embryos that do not contribute to future generations. Thus, in both Drosophila and mammals, the p53 gene family participates in an oocyte quality control system that protects the integrity of the transmitted genome (Chakravarti, 2022).

DNA ligase IV mutations confer shorter lifespan and increased sensitivity to nutrient stress in Drosophila melanogaster

The nonhomologous end-joining pathway is a primary DNA double-strand break repair pathway in eukaryotes. DNA ligase IV (Lig4) catalyzes the final step of DNA end ligation in this pathway. Partial loss of Lig4 in mammals causes Lig4 syndrome, while complete loss is embryonically lethal. DNA ligase 4 (DNAlig4) null Drosophila melanogaster is viable, but sensitive to ionizing radiation during early development. It is proposed to explore if DNAlig4 loss induced other long-term sensitivities and defects in D. melanogaster. This study demonstrated that DNAlig4 mutant strains had decreased lifespan and lower resistance to nutrient deprivation, indicating Lig4 is required for maintaining health and longevity in D. melanogaster (Joshi, 2022).

Usp5, Usp34, and Otu1 deubiquitylases mediate DNA repair in Drosophila melanogaster

Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, the direct repeat (DR)-white reporter system with which site-specific DSBs were induced was applied and the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways were applied using Drosophila. The results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process (Pahi, 2022).

The Drosophila Mutagen-Sensitivity Gene mus109 Encodes DmDNA2. Genes (Basel) 13(2).

The identification of mutants through forward genetic screens is the backbone of Drosophila genetics research, yet many mutants identified through these screens have yet to be mapped to the Drosophila genome. This is especially true of mutants that have been identified as mutagen-sensitive (mus), but have not yet been mapped to their associated molecular locus. This study addressed the need for additional mus gene identification by determining the locus and exploring the function of the X-linked mutagen-sensitive gene mus109 using three available mutant alleles: mus109(D1), mus109(D2), and mus109(lS). After first confirming that all three mus109 alleles were sensitive to methyl methanesulfonate (MMS) using complementation analysis, deletion mapping to narrow the candidate genes for mus109. Through DNA sequencing, it was possible to determine that mus109 is the uncharacterized gene CG2990, which encodes the Drosophila ortholog of the highly conserved DNA2 protein that is important for DNA replication and repair. The sequence and structure of DNA2 was used to predict the impact of the mus109 allele mutations on the final gene product. Together, these results provide a tool for researchers to further investigate the role of DNA2 in DNA repair processes in Drosophila (Mitchell, 2022).

CSB-independent, XPC-dependent transcription-coupled repair in Drosophila

An early study reported that Drosophila lacks the transcription-coupled repair (TCR) form of nucleotide excision repair. This conclusion was seemingly supported by the Drosophila genome sequencing project, which revealed that Drosophila lacks a homolog to CSB, which is known to be required for TCR in mammals and yeasts. However, by using excision repair sequencing (XR-seq) genome-wide repair mapping technology, it was recently found that the Drosophila S2 cell line performs TCR comparable to human cells. This study extended this work to Drosophila at all its developmental stages. It as find TCR takes place throughout the life cycle of the organism. Moreover, it was found that in contrast to humans and other multicellular organisms previously studied, the XPC repair factor is required for both global and transcription-coupled repair in Drosophila (Deger, 2022).

Cas9/Nickase-induced allelic conversion by homologous chromosome-templated repair in Drosophila somatic cells

Repair of double-strand breaks (DSBs) in somatic cells is primarily accomplished by error-prone nonhomologous end joining and less frequently by precise homology-directed repair preferentially using the sister chromatid as a template. In this study, a Drosophila system performs efficient somatic repair of both DSBs and single-strand breaks (SSBs) using intact sequences from the homologous chromosome in a process referred to as homologous chromosome-templated repair (HTR). Unexpectedly, HTR-mediated allelic conversion at the white locus was more efficient (40 to 65%) in response to SSBs induced by Cas9-derived nickases D10A or H840A than to DSBs induced by fully active Cas9 (20 to 30%). Repair phenotypes elicited by Nickase versus Cas9 differ in both developmental timing (late versus early stages, respectively) and the production of undesired mutagenic events (rare versus frequent). Nickase-mediated HTR represents an efficient and unanticipated mechanism for allelic correction, with far-reaching potential applications in the field of gene editing (Roy, 2022).

Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates

Human GEN1 and yeast Yen1 are endonucleases with the ability to cleave Holliday junctions (HJs), which are proposed intermediates in recombination. In vivo, GEN1 and Yen1 function secondarily to Mus81, which has weak activity on intact HJs. This study shows that the genetic relationship is reversed in Drosophila, with Gen mutants having more severe defects than mus81 mutants. In vitro, DmGen, like HsGEN1, efficiently cleaves HJs, 5 flaps, splayed arms, and replication fork structures. This study found that the cleavage rates for 5 flaps are significantly higher than those for HJs for both DmGen and HsGEN1, even in vast excess of enzyme over substrate. Kinetic studies suggest that the difference in cleavage rates results from a slow, rate-limiting conformational change prior to HJ cleavage: formation of a productive dimer on the HJ. Despite the stark difference in vivo that Drosophila uses Gen over Mus81 and humans use MUS81 over GEN1, in vitro activities of DmGen and HsGEN1 were found to be strikingly similar. These findings suggest that simpler branched structures may be more important substrates for Gen orthologs in vivo, and highlight the utility of using the Drosophila model system to further understand these enzymes (Bellendir, 2017).

Initiation of Drosophila chorion gene amplification requires Claspin and mus101, whereas Claspin, but not mus101, plays a major role during elongation

Claspin and TopBP1 are checkpoint mediators that are required for the phosphorylation of Chk1 by ATR to maintain genomic stability. This study investigated the functions of Drosophila Claspin and mus101 (TopBP1 ortholog) during chorion(eggshell component) gene amplification, which occurs in follicle cells. Unlike Drosophila mei-41 (ATR ortholog) mutant embryos, Claspin and mus101 mutant embryos showed severe eggshell defects resulting from defects in chorion gene amplification. EdU incorporation assay during initiation and elongation stages revealed that Claspin and mus101 were required for initiation, while only Claspin had a major role in the efficient progression of the replication forks. Claspin proteins were enriched in the amplification foci both in the initiation and elongation stage-follicle cell nuclei in a mei-41-independent manner. It is concluded that Drosophila Claspin plays a major role in the initiation and elongation stages of chorion gene amplification by localizing to the amplification foci in a mei-41-independent manner. Drosophila mus101 is also involved in chorion gene amplification, mostly functioning in initiation, rather than elongation (Choi, 2017).

To maintain genomic stability, the ATR and Chk1 checkpoint kinases play major roles in the DNA damage checkpoint response, which is induced by various types of DNA damage, including DNA replication stress. DNA replication stress activates these checkpoint genes, leading to inhibition of mitotic entry and stabilization of the replication fork to prevent fork collapse. Claspin and TopBP1 are checkpoint mediators that enhance ATR activity. In addition to their checkpoint functions, Chk1, Claspin, and TopBP1 are involved in normal DNA replication. The importance of the ATR, Chk1, Claspin, and TopBP1 genes during normal cell cycle progression is underscored by the embryonic lethality that results from mutations in these genes in mice. Drosophila contains the mei-41, Claspin, mus101, and grp genes, which are orthologs of ATR, Claspin, TopBP1, and Chk1, respectively. Studies of Drosophila Claspin mutants have demonstrated the involvement of Claspin in a replication stress-induced checkpoint during the midblastula transition, after hydroxyurea feeding, and in response to defective tRNA processing. Although the functions of Claspin during the checkpoint response have been extensively studied, its role during normal development is not well understood (Choi, 2017).

In the Drosophila ovary, somatic follicle cells encircle 16 germline cells, including the oocyte, and various cell cycle events occur in these follicle cells depending on their developmental stages. In addition to mitotic division, atypical cell cycle events, such as endoreplication and specific gene amplification in the absence of genomic replication, occur in somatic follicle cells during Drosophila oogenesis. During early development up to stage 6, follicle cells increase in number by undergoing mitotic divisions. Between stages 7 and 9, these cells endocycle by alternating between the S and gap phases. At stage 10, they cease genomic replication, and re-replication occurs from specific replication origins to amplify up to 60 copies of the chorion gene. The initiation and elongation stages of chorion gene replication occur during separate developmental stages of follicle cells; initiation occurs during stages 10B and 11, whereas only elongation from existing replication forks takes place during stages 12 and 13 (Choi, 2017).

Chorion is a major component of the eggshell and defects in chorion gene amplification result in a thin eggshell phenotype. Re-replication of the chorion gene induces DNA double-strand breaks, replication stress, and fork collapse, which is inhibited by mei-41, mus101, and grp to achieve efficient fork progression. The mus101 mutant embryo shows a thin eggshell phenotype due to defects in chorion gene amplification, while the grp mutant has a normal chorion gene copy number in amplification-stage follicle cells. However, the role of Claspin in chorion gene amplification is unknown. This study investigated the functions of Drosophila Claspin during chorion gene amplification and compared them with the functions of mei-41 and mus101 (Choi, 2017).

Drosophila Claspin and mus101 mutant embryos were found to show thin eggshell phenotypes due to reductions in chorion gene amplification, while mei-41 mutant embryos do not show obvious defects in chorion gene amplification. The chorion gene amplification detected by thymidine analog incorporation was greatly affected by Claspin mutations in both initiation- and elongation-stage follicle cells. Although initiation was significantly reduced in the mus101 mutant, the progression of replication forks in the elongation stage was not severely affected. The Claspin protein was enriched in chorion gene amplification foci during the initiation and elongation stages of chorion re-replication in a mei-41-independent manner. These results suggest that Drosophila Claspin and mus101 have a mei-41-independent function in the initiation of chorion gene amplification and Claspin, but not mus101, is important for the efficient progression of replication forks (Choi, 2017).

To understand the biological functions of Drosophila Claspin, this study investigated the basis of the thin eggshell phenotype of Claspin mutants and compared it with that of mus101 and mei&-41 mutants. Drosophila Claspin and mus101 were found to be required for the initiation of chorion gene amplification. Claspin, but not mus101, plays a major role in the efficient progression of replication forks. The role of Claspin during amplification was supported by its localization to amplification foci during initiation and elongation. These characteristics were distinct from those of mei&-41, suggesting that Drosophila Claspin and mus101 have a unique and mei&-41&-independent role in DNA replication during chorion gene amplification (Choi, 2017).

During oogenesis, the mode of DNA replication in somatic follicle cells that encircle germline cells changes from mitotic replication to endoreplication, followed by chorion gene amplification in the absence of genomic DNA replication. Studies of various mutants that show defects in chorion gene amplification have revealed three different phenotypes. In addition to a lack of amplification, some mutants exhibit chorion gene overamplification, and other mutant follicle cells fail to exit the endocycle during the amplification stage and instead perform inappropriate genomic DNA replication throughout the follicle cell genome. These results suggest that distinct signaling pathways exist for the positive and negative regulation of chorion gene amplification and for the repression of genomic DNA replication. In Claspin and mus101 mutant stage 10B follicle cells, neither ectopic genomic replication nor overamplification of the chorion gene was observed. This suggests that Claspin and mus101 are required for chorion gene amplification and that they are not involved in suppressing genomic DNA replication or in negatively regulating chorion gene amplification (Choi, 2017).

The functions of Claspin and TopBP1 in DNA replication are conserved from yeast to mammalian cells and both proteins are important for the initiation of DNA replication. This study found that Drosophila Claspin and mus101 are required for the initiation of chorion gene amplification based on the following observations. First, the intensity of EdU incorporation in follicle cells at the initiation stage and the relative fold amplification of ACE3, which is located 1.5 kb away from the origin, were severely reduced in both mutants. Second, when the EdU double bar was detected in the stage 13 follicle cells of Claspin and mus101 mutants, the length of the bar representing the number of origin firings was significantly shorter than that of the wild type . Lastly, the Claspin protein exhibited a focal localization overlapping with the largest EdU foci known to contain the ORC complex during the initiation stage (Choi, 2017).

In addition to initiation, Claspin affects the replication fork progression rate in mammalian cells (Petermann, 2008) and Mrc1 (yeast Claspin) found in the replisome is essential for rapid replisome progression in vitro. On the other hand, Dpb11 (yeast TopBP1) is not considered part of the replisome and Xenopus TopBP1 does not seem to be required for the elongation steps of DNA replication. Consistent with these previous reports, this study found that EdU foci were not efficiently resolved into a double bar structure in the Claspin mutant follicle cells at the elongation&-only stage, whereas a significantly higher percentage of mus101 mutant follicle cells exhibited double bar structure formation. Moreover, Claspin staining appeared as a double bar and colocalized with EdU during the elongation stage in follicle cells, visually confirming that Claspin moves along with the replication forks. These results show that Drosophila Claspin and mus101 have conserved functions during chorion gene amplification (Choi, 2017).

Drosophila chorion gene amplification begins with the binding of the ORC complex to replication origins using most of the general DNA replication machinery. Many genes have been reported to affect chorion gene amplification and mutations in most of these genes also result in a loss of ORC foci formation. The exceptions are Myb and dup mutants; normal ORC2 foci have been detected, despite the absence of bromodeoxyuridine foci in the Myb mutant clones and ORC2 foci are smaller in dup mutant follicle cells (Choi, 2017).

This study found that ORC2 localization to amplification loci was significantly reduced in the mus101 K451 mutant compared with the wild type, whereas it was not significantly different in Claspin 45 mutant. Compared with the wild type, the amplification of ACE3 in Claspin 45 and mus101 K451 mutants was reduced to 24.0 and 5.2&-fold relative to actin, respectively. Because ACE3 is the region recognized by ORC2 and where the major ORC2 foci are localized at stage 10B, a significant reduction in ORC2 intensity in the mus101 mutant is likely to result from the reduced copy number of the origin (Choi, 2017).

Additionally, the Dup (Drosophila Cdt1) protein, which usually forms foci at chorion loci, is stabilized and delocalized by various defects in DNA replication, including mus101 K451 mutations. It is not clear if Dup localization is similarly affected in Claspin mutants. Because the size of ORC2 foci is smaller in dup mutant follicle cells than in wild type cells, the reduction in ORC2 intensity found in mus101 K451 mutants may result from the delocalization of Dup. Further analyses will be required to elucidate the detailed molecular events in the initiation steps of chorion gene amplification (Choi, 2017).

A previous study of Drosophila mei&-41 RT1 and mus101 D1, a separation-of-function allele that shows defects in the G2/M DNA damage checkpoint, but normal DNA replication, showed that cells lacking these genes are defective in the replication stress checkpoint and exhibit reduced fork progression by 25%-30%, rather than the complete lack of replication. mus101 K451, another separation&-of&-function allele with the opposite phenotypes, shows defects mostly in the initiation step of chorion gene amplification. Claspin is directly involved in the initiation and elongation steps of chorion gene amplification, although mitotic replication and endoreplication seem to occur normally in both mutants. Because several hypomorphic mutants of pre&-RC components also show phenotypic abnormalities only in chorion amplification, amplification may be more sensitive to the activity of the basal DNA replication machinery than to mitotic replication (Choi, 2017).

A recent study reporting the first example of gene amplification in normal mammalian development has identified genes that are selectively amplified in trophoblast giant cells. An investigation into whether the Claspin and TopBP1 play similar functions in mammals will provide useful insights. Drosophila chorion gene amplification will serve as a valuable model for elucidating the mechanism of action of Claspin and mus101 during DNA replication, specific gene amplification, and the replication stress checkpoint (Choi, 2017).

Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation

Mismatch repair (MMR) system, a conserved DNA repair pathway, plays crucial role in DNA recombination and is involved in gametogenesis. This study analysed the impact of mlh1 (a MutL homologue) on meiotic crossing over/recombination and fertility in a genetically tractable model, Drosophila melanogaster. Using mlh1e00130 hypomorphic allele, this study reports female specific adverse reproductive outcome for reduced mlh1 in Drosophila: mlh1e00130 homozygous females had severely reduced fertility while males were fertile. Further, mlh1e00130 females contained small ovaries with large number of early stages as well as significantly reduced mature oocytes, and laid fewer eggs, indicating discrepancies in egg production and ovulation. These observations contrast the sex independent and/or male specific sterility and normal follicular development as well as ovulation reported so far for MMR family proteins in mammals. However, analogous to the role(s) of mlh1 in meiotic crossing over and DNA repair processes underlying mammalian fertility, ovarian follicles from mlh1e00130 females contained significantly increased DNA double strand breaks (DSBs) and reduced synaptonemal complex foci. In addition, large proportion of fertilized eggs display discrepancies in egg activation and fail to proceed beyond stage 5 of embryogenesis. Hence, reduction of the Mlh1 protein level leads to defective oocytes that fail to complete embryogenesis after fertilization thereby reducing female fertility (Vimal, 2017).

Nuclear F-actin and myosins drive relocalization of heterochromatic breaks

Heterochromatin mainly comprises repeated DNA sequences that are prone to ectopic recombination. In Drosophila cells, 'safe' repair of heterochromatic double-strand breaks by homologous recombination relies on the relocalization of repair sites to the nuclear periphery before strand invasion. The mechanisms responsible for this movement were unknown. This study shows that relocalization occurs by directed motion along nuclear actin filaments assembled at repair sites by the Arp2/3 complex. Relocalization requires nuclear myosins associated with the heterochromatin repair complex Smc5/6 and the myosin activator Unc45, which is recruited to repair sites by Smc5/6. ARP2/3, actin nucleation and myosins also relocalize heterochromatic double-strand breaks in mouse cells. Defects in this pathway result in impaired heterochromatin repair and chromosome rearrangements. These findings identify de novo nuclear actin filaments and myosins as effectors of chromatin dynamics for heterochromatin repair and stability in multicellular eukaryotes (Caridi, 2018).

Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A

Repair of DNA double-strand breaks (DSBs) must be orchestrated properly within diverse chromatin domains in order to maintain genetic stability. Euchromatin and heterochromatin domains display major differences in histone modifications, biophysical properties, and spatiotemporal dynamics of DSB repair. However, it is unclear whether differential histone-modifying activities are required for DSB repair in these distinct domains. Previous work has shown that the Drosophila melanogaster KDM4A (dKDM4A) histone demethylase is required for heterochromatic DSB mobility. This study used locus-specific DSB induction in Drosophila animal tissues and cultured cells to more deeply interrogate the impact of dKDM4A on chromatin changes, temporal progression, and pathway utilization during DSB repair. dKDM4A was found to promote the demethylation of heterochromatin-associated histone marks at DSBs in heterochromatin but not euchromatin. Most importantly, it was demonstrated that dKDM4A is required to complete DSB repair in a timely manner and regulate the relative utilization of homologous recombination (HR) and nonhomologous end-joining (NHEJ) repair pathways but exclusively for heterochromatic DSBs. It is concluded that the temporal kinetics and pathway utilization during heterochromatic DSB repair depend on dKDM4A-dependent demethylation of heterochromatic histone marks. Thus, distinct pre-existing chromatin states require specialized epigenetic alterations to ensure proper DSB repair (Janssen, 2019).

Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster

Meiotic recombination, which is necessary to ensure that homologous chromosomes segregate properly, begins with the induction of meiotic DNA double-strand breaks (DSBs) and ends with the repair of a subset of those breaks into crossovers. This study investigated the roles of two paralogous genes, CG12200 and CG31053, which have been named Narya and Nenya, respectively, due to their relationship with a structurally similar protein named Vilya. narya recently evolved from nenya by a gene duplication event, and these two RING finger domain-containing proteins were shown to be functionally redundant with respect to a critical role in DSB formation. Narya colocalizes with Vilya foci, which are known to define recombination nodules, or sites of crossover formation. A separation-of-function allele of narya retains the capacity for DSB formation but cannot mature those DSBs into crossovers. Data is provided on the physical interaction of Narya, Nenya and Vilya, as assayed by the yeast two-hybrid system. Together these data support the view that all three RING finger domain-containing proteins function in the formation of meiotic DNA DSBs and in the process of crossing over (Lake, 2019).

A robust transposon-endogenizing response from germline stem cells

The heavy occupancy of transposons in the genome implies that existing organisms have survived from multiple, independent rounds of transposon invasions. However, how and which host cell types survive the initial wave of transposon invasion has remained unclear. This study shows that the germline stem cells can initiate a robust adaptive response that rapidly endogenizes invading P element transposons by activating the DNA damage checkpoint and piRNA production. Temperature modulates the P element activity in germline stem cells, establishing a powerful tool to trigger transposon hyper-activation. Facing vigorous invasion, Drosophila first shut down oogenesis and induce selective apoptosis. Interestingly, a robust adaptive response occurs in ovarian stem cells through activation of the DNA damage checkpoint. Within 4 days, the hosts amplify P element-silencing piRNAs, repair DNA damage, subdue the transposon, and reinitiate oogenesis. It is proposed that this robust adaptive response can bestow upon organisms the ability to survive recurrent transposon invasions throughout evolution (Moon, 2018).

Considered as 'selfish DNA sequences,' transposons have heavily accumulated in the genome of nearly all organisms during evolution. Although capable of fueling genomic divergence, the transposon invasion process is disruptive to host cells and often severely impacts host fertility or even survival. Therefore, taming invading transposons is an essential and endless task for the host organism. In this study, by using P element invasion as a model, temperature shifting was established as a powerful tool to adjust the intensity of transposon invasion. By investigating the response from the Drosophila adult ovaries, in which P element activity and germ cell development can be measured in detail, a robust transposon-endogenizing mechanism from the germline stem cells was uncovered. Centered on the key DNA damage checkpoint component, Chk2, this robust adaptive response renders hosts the ability to permanently silence invading transposons within just 4 days (Moon, 2018).

GFP::Vasa mobilization assay shows that the P element actively hops in germline stem cells. Does the P element also mobilize in other ovarian cells? Since nurse cells are polyploid and the developing oocytes are transcriptionally inactive, the current assay could not faithfully monitor P element mobilization in them. However, previous study shows that nurse cells express the protein P-element somatic inhibitor (PSI), which can block intron removal of P element transcripts and lead to the production of inactive transposases. Therefore, it is unlikely that P elements mobilize within developing egg chambers. As a type of DNA transposon, which employs the cut-and-paste mechanism for transposition, P elements cannot directly increase their copy number through mobilization. Instead, the propagation is likely achieved via homologous repair from the sister DNA strand during S-phase of the cell cycle. Hence, to amplify themselves during Drosophila oogenesis, perhaps P elements evolved to preferentially mobilize in the dividing germline stem cells but not in the developing oocytes, which are under cell cycle arrest (Moon, 2018).

By investigating adult oogenesis of Drosophila, this study uncovered the Chk2-mediated adaptive response from germline stem cells upon P element transposon invasion (Moon, 2018).

Interestingly, it appears that arrested germ cells are not equally capable of taming transposons, and Chk2 activation promotes adaptation by eliminating the cells with lower competency. Several lines of evidence support the occurrence of selective cell elimination. First, a significant increase in cell death was detected once P elements became hyperactive after the temperature shift. Second, although GFP-negative egg chambers directly connected to germaria at 25°C were occasionally observed from the GFP::Vasa mobilization assay, no GFP-negative cells were detected in later stage egg chambers at any time points. This suggests that the germ cells that maintained high P element activity, and were presumably less competent to adapt, were eliminated at early stages of oogenesis. Third, the number of new P element insertion events declined to 44% in recovered ovaries after adaptation. This dramatic decline indicates that only the stem cells that had lower transposition rates survived the selection. Therefore, it is tempting to speculate that not all germ cells are created equal and that in addition to germarial arrest, the Chk2-mediated DNA break checkpoint also has a role in selecting the survivors from P element invasion and promoting adaptation (Moon, 2018).

In the surviving ovarian stem cells, Chk2-mediated oogenesis arrest provides a critical time window to propel piRNA generation from the paternally inherited clusters, initiating the amplification cycles for piRNA biogenesis. With at least two piRNA clusters containing P element sequences in the paternally inherited genome, invaded progeny are capable of generating P element-silencing piRNAs de novo. Although it is still unclear when the clusters become active during pre-adult development, it has been shown that the primordial germ cells in larval ovaries can already initiate de novo piRNA production. Consistently, low levels of piRNAs were detected corresponding to P element before adaptation. However, it appears that the amount of piRNAs produced at this stage is too scarce to silence invading P elements. Their activation results in sterility and triggers the Chk2-dependent acute adaptive response from germline stem cells. Subsequently, the Chk2-mediated arrest blocks differentiation, which would allow the newly produced P element-silencing piRNAs to quickly reach a concentration sufficient for Ping-Pong amplification. Finally, these newly produced piRNAs silence transposons at the post transcriptional level and also initiate transcriptional silencing (Moon, 2018).

Besides promoting piRNA production, the arrest period also allows germ cells to repair DNA lesions before reinitiating oogenesis, thereby preventing the proliferation of cells with DNA damage and defective differentiation. Having the ability to repair damage and endogenize invading transposons in germline stem cells ensures permanent restoration of robust oogenesis and protection of all daughter cells from transposon activation (Moon, 2018).

Heterochromatic breaks move to the nuclear periphery to continue recombinational repair

Heterochromatin mostly comprises repeated sequences prone to harmful ectopic recombination during double-strand break (DSB) repair. In Drosophila cells, 'safe' homologous recombination (HR) repair of heterochromatic breaks relies on a specialized pathway that relocalizes damaged sequences away from the heterochromatin domain before strand invasion. This study shows that heterochromatic DSBs move to the nuclear periphery to continue HR repair. Relocalization depends on nuclear pores and inner nuclear membrane proteins (INMPs) that anchor repair sites to the nuclear periphery through the Smc5/Smc6-interacting proteins STUbL/RENi. Both the initial block to HR progression inside the heterochromatin domain, and the targeting of repair sites to the nuclear periphery, rely on SUMO and SUMO E3 ligases. This study reveals a critical role for SUMOylation in the spatial and temporal regulation of HR repair in heterochromatin, and identifies the nuclear periphery as a specialized site for heterochromatin repair in a multicellular eukaryote (Ryu, 2015).

Nuclear architecture contributes to HR repair of certain types of DSBs in budding yeast. Specifically, most DSBs exhibit Brownian motion and remain in the nucleoplasm during HR, but persistent DSBs are shunted to the nuclear periphery after resection. This relocalization has been observed in conditions where HR repair is effectively stalled, such as in the absence of a donor sequence for repair or after fork collapse. Whether relocalization is a physiological response to DSBs is still controversial, and the existence of similar roles for the nuclear periphery in multicellular eukaryotes has not been addressed (Ryu, 2015).

Pericentromeric heterochromatin occupies about 30% of fly and human genomes and is characterized by large contiguous stretches of repeated sequences (transposons and 'satellite' repeats) and the 'silent' epigenetic marks H3K9me2/3 and Heterochromatin Protein 1 (HP1a in Drosophila). While pericentromeric heterochromatin is absent in budding yeast, it represents a major threat to genome stability in multicellular eukaryotes. Thousands to millions of identical repeated sequences on different chromosomes can engage in ectopic recombination and generate chromosome rearrangements (e.g., acentric and dicentric chromosomes) during DSB repair. Previous work has identified a mechanism that promotes HR repair while preventing aberrant recombination in Drosophila. Early HR steps (resection and ATRIP/TopBP1 recruitment) occur quickly within the heterochromatin domain, but later steps (Rad51 recruitment) occur only after repair sites have relocalized to outside the domain. Relocalization of heterochromatic DSBs also occurs in mouse cells, suggesting that this mechanism is conserved. It is proposed that relocalization prevents aberrant recombination by separating damaged DNA from similar repeats on non-homologous chromosomes, while promoting 'safe' exchanges with the sister chromatid or homolog. Removing heterochromatic proteins (e.g., Smc5/6) results in relocalization defects, abnormal recruitment of Rad51 inside the heterochromatin domain, and massive aberrant recombination between heterochromatic sequences, revealing the importance of this pathway to genome stability. Whether heterochromatic DSBs relocalize to a specific subnuclear compartment was unclear, and the mechanisms responsible for relocalization and the regulation of HR progression were unknown (Ryu, 2015).

These studies reveal the nuclear periphery as a specialized site for repairing heterochromatic DSBs in Drosophila. DSBs leave the heterochromatin domain and relocalize to nuclear pores or INMPs to continue HR repair, and this process is mediated by STUbL/RENi proteins associated with these nuclear periphery components. This study identified the Nup107-160 sub-complex and Koi and Spag4 INMPs as specific anchoring sites for the STUbL/RENi complex Dgrn/dRad60 and for repair sites. Further, recruitment of dRad60 to the nuclear periphery relies on Dgrn, and both physically associate with Smc5/6 in response to damage. This suggests that interactions between Smc5/6 and Dgrn/dRad60 stabilize the association of heterochromatic DSBs with the nuclear periphery. Finally, Nse2 and dPIAS SUMO ligases and SUMO are required for both relocalizing DSBs and preventing Rad51 recruitment inside the heterochromatin domain (Ryu, 2015).

It is proposed that SUMOylation of one or more HR components after resection, generates a temporary block to Rad51 recruitment inside the heterochromatin domain to prevent ectopic recombination. Relocalization to the nuclear periphery isolates the broken DNA, presumably together with its homologous template (sister chromatid and/or homolog) to complete 'safe' repair. STUbL might mediate the removal of this block by ubiquitylating poly-SUMOylated components, and inducing their proteasome-mediated degradation or recognition by other repair proteins. Potential SUMOylated targets include histones, RPA, Mdc1/Mu2, Smc5/6 subunits, Blm, and other repair and heterochromatin components. Inactivation of this pathway causes instability of repeated sequences and chromosome aberrations, revealing its critical role in heterochromatin repair and genome integrity. Importantly, inactivation of this pathway also leads to disrupted micronuclei, potentially contributing to DNA damage and genome instability in cancer cells (Ryu, 2015).

Aspects of this pathway are surprisingly similar to the mechanism that targets persistent DSBs to the nuclear periphery in S. cerevisiae, including the role of Smc5/6 and SUMO. This likely results from common signaling mechanisms, such as SUMOylation of repair components following extensive resection. However, this similarity is unexpected because budding yeast lacks the long stretches of pericentromeric repeats that present a major challenge for DSB repair in Drosophila and human cells, as well as H3K9 methylation and HP1 proteins that are required for spatial and temporal regulation of heterochromatic HR repair. Remarkably, a pathway utilized by yeast to deal with a rare class of 'persistent' DSBs, collapsed forks, or eroded telomeres, is now emerging as one of the most important mechanisms to safeguard genome stability in multicellular eukaryotes (Ryu, 2015).

Homolog dependent repair following dicentric chromosome breakage in Drosophila melanogaster

Double strand DNA breaks are repaired by one of several mechanisms that rejoin two broken ends. However, cells are challenged when asked to repair a single broken end, and respond by: (1) inducing programmed cell death; (2) healing the broken end by constructing a new telomere; (3) adapting to the broken end and resuming the mitotic cycle without repair; (4) using information from the sister chromatid or homologous chromosome to restore a normal chromosome terminus. During one form of homolog dependent repair (HDR) in yeast, termed Break Induced Replication (BIR), a template chromosome can be copied for 100s of kb. BIR efficiency depends on Pif1 helicase and Pol32, a non-essential subunit of DNA polymerase delta. To date, there is little evidence that BIR can be used for extensive chromosome repair in higher eukaryotes. This study reports that a dicentric chromosome broken in mitosis in the male germline of Drosophila melanogaster is usually repaired by healing, but can also be repaired in a homolog dependent fashion, restoring at least 1.3 Mb of terminal sequence information. This mode of repair is significantly reduced in pif1 and pol32 mutants. Formally, the repaired chromosomes are recombinants. However, the absence of reciprocal recombinants, and the dependence on Pif1 and Pol32, strongly support the hypothesis that BIR is the mechanism for restoration of the chromosome terminus. In contrast to yeast, pif1 mutants in Drosophila exhibit a reduced rate of chromosome healing, likely owing to fundamental differences in telomeres between these organisms (Bhandari, 2019).

Chromosome preference during homologous recombination repair of DNA double-strand breaks in Drosophila melanogaster

DNA double-strand breaks (DSBs) are especially toxic DNA lesions that, if left unrepaired, can lead to wide-ranging genomic instability. Of the pathways available to repair DSBs, the most accurate is homologous recombination (HR), where a homologous sequence is used as a donor template to restore genetic information at the break site. While much of the biochemical aspects of HR repair have been characterized, how the repair machinery locates and discriminates between potential homologous donor templates throughout the genome remains elusive. This study used Drosophila melanogaster to investigate whether there is a preference between intrachromosomal and interhomolog donor sequences in mitotically dividing cells. The results demonstrate that, although interhomolog HR is possible and frequent if another donor template is not available, intrachromosomal donor templates are highly preferred. This is true even if the interhomolog donor template is less diverged than the intrachromosomal donor template. Thus, despite the stringent requirements for homology, the chromosomal location of the donor template plays a more significant role in donor template choice (Fernandez, 2019).

Drosophila, which lacks canonical transcription-coupled repair proteins, performs transcription-coupled repair

Previous work with the classic T4 endonuclease V digestion of DNA from irradiated Drosophila cells followed by Southern hybridization led to the conclusion that Drosophila lacks transcription-coupled DNA repair (TCR). This conclusion was reinforced by the Drosophila Genome Project which revealed that Drosophila lacks Cockayne syndrome WD repeat protein (CSA), CSB, or UV-stimulated scaffold protein A (UVSSA) homologs, whose orthologs are present in eukaryotes ranging from Arabidopsis to humans that carry out TCR. A recently developed in vivo excision assay and the excision repair-sequencing (XR-Seq) method have enabled genome-wide analysis of nucleotide excision repair in various organisms at single-nucleotide resolution and in a strand-specific manner. Using these methods, it was discovered that Drosophila S2 cells carry out robust TCR comparable to that observed in mammalian cells. These findings provide critical new insights into the mechanisms of TCR among various different species (Deger, 2019).

X chromosome and autosomal recombination are differentially sensitive to disruptions in SC maintenance

The synaptonemal complex (SC) is a conserved meiotic structure that regulates the repair of double-strand breaks (DSBs) into crossovers or gene conversions. The removal of any central-region SC component, such as the Drosophila melanogaster transverse filament protein C(3)G, causes a complete loss of SC structure and crossovers. To better understand the role of the SC in meiosis, CRISPR/Cas9 was used to construct 3 in-frame deletions within the predicted coiled-coil region of the C(3)G protein. Since these 3 deletion mutations disrupt SC maintenance at different times during pachytene and exhibit distinct defects in key meiotic processes, they allowed definition the stages of pachytene when the SC is necessary for homolog pairing and recombination during pachytene. These studies demonstrate that the X chromosome and the autosomes display substantially different defects in pairing and recombination when SC structure is disrupted, suggesting that the X chromosome is potentially regulated differently from the autosomes (Billmyre, 2019).

Several facets of meiosis ensure the faithful inheritance of chromosomes from parents to offspring. During the creation of eggs and sperm the genome must be reduced to a haploid state containing a single set of chromosomes. The failure to properly segregate chromosomes results in gametes with an incorrect number of chromosomes. Indeed, errors in meiotic chromosome segregation are the leading cause of miscarriage and aneuploidy in humans, which can result in chromosomal disorders such as Down syndrome and Turner syndrome (Billmyre, 2019).

Proper segregation of chromosomes during meiosis relies on the formation of programmed double-strand breaks (DSBs), which are initiated by the evolutionarily conserved type II DNA topoisomerase-like protein Spo11 (Mei-W68 in Drosophila). These DSBs are then repaired as crossover or gene conversion events. Crossovers mature into chiasmata, which physically hold homologous chromosomes together from nuclear envelope breakdown until homolog separation at anaphase I, thus ensuring proper segregation of chromosomes. The placement of crossover events is highly nonrandom and is strictly regulated by multiple processes. First, crossover interference prevents 2 crossovers from occurring in close proximity to each other. Second, crossovers are excluded from the heterochromatin. Third, as a result of the centromere effect, crossing over is also reduced in those euchromatic regions that lie in proximity to the centromeres. Finally, even within the medial and distal euchromatin, crossing over is substantially higher toward the middle of the chromosome arms. These constraints do not affect the frequency or distribution of gene conversion events, which appear to be randomly distributed throughout the euchromatin. Thus, the control of crossover distribution may act at the level of DSB fate choice, rather than in determining the position of DSBs (Billmyre, 2019).

Previous studies have demonstrated that the synaptonemal complex (SC), a large protein structure that forms between homologous chromosomes, plays a role in controlling crossover distribution. The SC is a highly conserved tripartite structure, with 2 lateral elements and a central region. The central region is composed of transverse filament and central element proteins, while the lateral element proteins connect the central region to the chromosome axes. The known proteins that make up the Drosophila central region include the main transverse filament protein C(3)G, the transverse filament-like protein Corolla, and the central element protein Corona (CONA) (Billmyre, 2019).

Work in Caenorhabditis elegans has shown that the SC functions to monitor crossover placement by preventing additional crossover designation in a region adjacent to an existing crossover precursor. Furthermore, there is evidence in Saccharomyces cerevisiae that Zip1, a transverse filament protein, has 2 separable functions-one in building the SC and the other in recombination. Lastly, in rice, there is evidence that a partial loss of the SC results in increased crossing over and crossover proximity similar to what was reported in C. elegans. Based on what is known in other model systems, it is likely that the Drosophila SC is also playing a role in regulating the fate of DSBs and monitoring crossover placement (Billmyre, 2019).

In Drosophila females, ~24 DSBs are formed in early pachytene. Unlike in many other organisms where DSBs occur prior to SC formation, in Drosophila DSBs are formed in the context of fully formed SC. In the absence of the central region of the SC, DSB formation is substantially reduced, but not eliminated. Nonetheless, even in the presence of a substantial number of residual DSBs (37% of wild type), the loss of these SC proteins results in a complete loss of crossover formation. The abolishment of the central region of the SC also results in a high frequency of unpaired homologs during pachytene. In addition to disrupting meiotic pairing, the loss of any of the known central-region components in the (premeiotic) mitotic region of the ovaries also impairs mitotic pairing of the second and third chromosomes (Billmyre, 2019).

Since the vast majority of SC mutants in Drosophila are null mutants and therefore fail to form any SC structure, it is difficult to investigate the interactions of the wild-type versions of these proteins at the protein level or discover how the SC is involved in DSB repair and fate choice. In Drosophila, the study of transgenes carrying in-frame deletions of either the N- or C-terminal globular domains of C(3)G has shown that both of these regions are required for proper SC assembly and crossover formation. However, these defects were too severe to allow investigation of the function of the SC in crossover placement and formation. One domain which has not been tested is the large predicted coiled-coil domain in C(3)G. Coiled-coil domains are a key conserved feature of transverse filament proteins across many organisms and are known to be important for protein-protein interactions (Billmyre, 2019).

This study has characterize 3 in-frame deletion mutations in the coiled-coil domain of the Drosophila melanogaster c(3)G, all of which cause a partial loss of SC function at different stages in early meiosis. Advantage was taken of the different stages of SC loss to examine when the SC is necessary for multiple meiotic events such as pairing and recombination. Unlike any previously characterized Drosophila meiotic mutants, the effects of these mutants on X chromosome recombination is different from their effects on autosomal recombination. It is inferred from this observation that chromosomes can respond differently to a failure in SC maintenance. It was also shown that the SC in early pachytene is important for the maintenance of euchromatic pairing, especially in the distal euchromatin (in relation to the centromere) regions of the chromosome arms. The maintenance of X chromosome pairing is more sensitive to SC defects than is pairing maintenance on the autosomes, suggesting there may be additional chromosome-specific processes that mediate pairing. These mutants allowed us to examine the temporal requirement for the synaptonemal complex in crossover placement and maintenance of pairing (Billmyre, 2019).

Both the regulation of SC assembly and disassembly, and its maintenance after assembly, is poorly understood. Work in other organisms has shown that posttranslational modifications are important in SC structure and function. It is known that SUMOylation and N-terminal acetylation promote assembly of the SC while phosphorylation or dephosphorylation promote disassembly of the SC with modifications occurring on multiple SC proteins. Thus far, no posttranslationally modified sites have been identified on C(3)G. However, it is likely that these sites do exist, and it is speculated that sites promoting SC assembly, maintenance, and disassembly may be disrupted in these mutants (Billmyre, 2019).

Another possibility is that the deletions described in this study could destabilize protein-protein interaction sites between C(3)G and other central-region proteins, resulting in an unstable SC that is difficult to maintain. It is noted that the mutant with the smallest deletion, c(3)GccΔ3, exhibited the strongest SC defect. While this deletion was predicted to only disrupt a single coil, the best explanation for the more severe phenotype is that it actually disrupts the coiled coil. This may have caused a large disruption in the rest of the coiled-coil structure. In the future, it will be important to further dissect these domains to better understand the regulation of SC assembly and disassembly (Billmyre, 2019).

A surprising result from these studies was the ability of these deletions to allow the progressive loss of homologous euchromatic pairing through pachytene. The mechanism behind establishing and maintaining homolog pairing is a long-standing, unanswered question in the meiosis field. Previous work in Drosophila has shown that in the complete absence of the central-region proteins C(3)G and CONA, euchromatic pairing is significantly reduced in early to mid and mid pachytene (Billmyre, 2019).

Partial loss-of-function mutations have allowed testing of the importance of C(3)G in maintaining pairing throughout pachytene when the SC is present in early pachytene (unlike previous studies of null mutants in which the SC is always absent). From these mutants, a timeline is now available of when the SC is necessary to maintain pairing and recombination on the X chromosome and the autosomes. By comparing these mutants, it is hypothesized that the X chromosome needs a full-length SC earlier in pachytene for proper maintenance of pairing and recombination while the autosomes are likely capable of placing crossovers as late as mid pachytene, resulting in a proximal euchromatin shift in crossovers where pairing is maintained (Billmyre, 2019).

In both c(3)GccΔ1 and c(3)GccΔ3 mutants, distal pairing of the X chromosome and the autosomes was most strongly reduced. One likely explanation for this stronger effect on distal regions of the chromosome arms is that normally the disassembly of the SC is initiated on the euchromatic chromosome arms with the centromeric region being removed last. Since the loss of the SC in c(3)GccΔ1 and c(3)GccΔ3 mutants occurs in a manner similar to wild-type SC disassembly, the distal regions of the chromosome may be affected earlier and more strongly than the proximal euchromatin regions. The proximal euchromatin region contains a large amount of heterochromatin that could be mediating pairing interactions and stabilizing pairing in the absence of the SC. Furthermore, examination of centromere pairing suggests that the centromeres are still paired and could be facilitating the proximal euchromatic pairing. This idea is supported by the higher levels of proximal euchromatic pairing compared with distal pairing in c(3)Gnull (Billmyre, 2019).

Finally, it is speculated that the ability of the c(3)GccΔ1 mutants to exhibit a distal euchromatic pairing defect that is more severe than the defect seen in c(3)G null mutants results from the residual proximal crossovers that do form in c(3)GccΔ1 mutants. Previous work has shown that crossovers can preserve synapsis but only in their vicinity. Perhaps the stresses that provoke separation become more concentrated on the distal regions that lack crossovers. For example, it is possible that the untethered distal regions could experience a higher mechanical stress due to nuclear movements than the pericentric regions containing a crossover. The lack of a strong pairing defect in c(3)GccΔ2 mutants is probably due to the persistence of a full-length SC until mid pachytene. Together, these data support a role for the SC in maintaining euchromatic pairing during early to mid prophase (Billmyre, 2019).

The autosomal increase in proximal euchromatin crossovers displayed in these mutants mimics the interchromosomal effect. The interchromosomal effect has been reported in flies that are heterozygous for chromosome aberrations that suppress exchange in trans to a wild-type chromosome. Thus, the absence of crossover formation on one chromosome promotes increased recombination on the other chromosomes, with more crossovers placed in the proximal euchromatin regions. The mechanism that controls the interchromosomal effect in balancer heterozygotes is poorly understood. Additionally, the interchromosomal effect has been reported in C. elegans mutants with defective synapsis, further supporting this possibility (56). It is possible that the interchromosomal effect is partially responsible for the increase in proximal euchromatin crossovers in c(3)GccΔ1 and c(3)GccΔ3 mutants due to the loss of X chromosome recombination (Billmyre, 2019).

However, the interchromosomal effect cannot explain the increase in proximal euchromatin recombination in c(3)GccΔ2 mutants since X recombination appears normal. In theory, this phenotype could be explained by crossover homeostasis, which functions to control the number of crossovers so the appropriate number is placed. In many organisms, when there is a deficit of crossovers by the end of early to mid pachytene, the cell will continue to place crossovers in alternative locations to maintain an appropriate number. Such a process could result in crossovers being placed later than normal, which could be an issue when the SC is breaking down prematurely and homolog pairing is lost. However, Mehrotra and McKim (2006) provide evidence that crossover homeostasis is unlikely to occur in Drosophila females. It is unknown how much of a role the SC plays in the repair of DSBs into crossover versus non-crossover events. It is possible the SC must be present to interact with factors necessary for regulating the placement of crossovers. For example, Vilya, a pro-crossover factor, localizes to the SC and DSBs prior to being recruited to recombination nodules. If DSB repair on the autosomes does not occur until early to mid pachytene and the SC is necessary for the determination of a crossover fate, it follows that loss of the SC in the euchromatin would result in a shift of crossover formation toward proximal euchromatin regions where the SC may still be present. This mechanism could also be increasing proximal euchromatin recombination in c(3)GccΔ1 and c(3)GccΔ3 flies. Alternatively, SC-independent heterochromatic pairing may be holding the proximal euchromatin region in close proximity, allowing for crossing over in that region. In addition to interacting with pro-crossover factors, the SC may be interacting with a currently unknown protein which regulates crossover placement differently on the X chromosome versus the autosomes (Billmyre, 2019).

This set of mutants represents a unique tool to investigate not only the temporal requirements of the SC but also the differences in crossover placement between the X chromosome and the autosomes. Since c(3)GccΔ2 mutants do not display defects in X chromosome recombination, it is concluded that a full-length SC throughout early to mid pachytene is sufficient for X chromosome crossover placement but not for normal distribution of autosomal crossovers. Examining autosomal recombination in all 3 mutants suggests that a full-length SC is necessary in mid pachytene for proper crossover distribution on the autosomes. There are multiple explanations for the recombination differences between the X chromosome and the autosomes (Billmyre, 2019).

The first of these hypotheses is that there might exist a timing difference in either synapsis or crossover placement between the X chromosome and the autosomes. Work in C. elegans has provided evidence for timing differences between the sex chromosomes and the autosomes. For example, the X chromosome initiates premeiotic DNA replication later than the autosomes. This could be significant, as replication timing has been shown to impact crossover designation in barley. Additionally, in C. elegans, the X chromosome and the autosomes pair at the same time, but synapsis of the X chromosome is delayed and the X chromosome has lower levels of DSB formation compared with the autosomes (58, 61). Thus, the timing of when each chromosome is fully synapsed could be critical to ensure normal crossover placement, and the premature disruption of synapsis may affect the activity of pro-crossover factors. For example, in C. elegans, the XND-1 protein is required for genome-wide crossover placement and is important for normal rates of DSBs on the X chromosome. Currently, it is unknown in Drosophila if there are differences in the timing of DSB repair or synapsis of the X chromosome as compared with the autosomes, and the data suggest this as a possibility (Billmyre, 2019).

A second, but not mutually exclusive, explanation for the differences between the chromosomes may be a structural one. The X chromosome is acrocentric (the centromere is near the end of the chromosome), while the autosomes are both metacentric (the centromere is near the center of the chromosome) and, perhaps, these structural differences mean that the X chromosome is more sensitive to loss of the SC. The data suggest that loss of SC maintenance disrupts the maintenance of euchromatic homolog pairing more severely on the X chromosome than on the autosomes. It is unknown if metacentric chromosomes are different in terms of synapsis and recombination as compared with acrocentric chromosomes, and further investigation is needed to determine if structural differences affect these processes (Billmyre, 2019).

It is clear from decades of research that the regulation of recombination requires many factors and precise timing. This study shows that the SC plays a vital role in maintaining homolog pairing and proper crossover distribution in Drosophila female meiosis. Many differences between sex chromosomes and autosomes have been documented in a multitude of organisms, and our data are consistent with these differences extending into the processes that control chromosome pairing and recombination. With this set of mutants, a system has been established to examine X chromosome and autosome biology in Drosophila meiosis that will allow future work to unravel the mechanism behind meiotic chromosomal differences (Billmyre, 2019).

Synaptonemal complex-deficient Drosophila melanogaster females exhibit rare DSB repair events, recurrent copy-number variation, and an increased rate of de novo transposable element movement

Genetic stability depends on the maintenance of a variety of chromosome structures and the precise repair of DNA breaks. During meiosis, programmed double-strand breaks (DSBs) made in prophase I are normally repaired as gene conversions or crossovers. DSBs can also be made by other mechanisms, such as the movement of transposable elements (TEs), which must also be resolved. Incorrect repair of these DNA lesions can lead to mutations, copy-number changes, translocations, and/or aneuploid gametes. In Drosophila melanogaster, as in most organisms, meiotic DSB repair occurs in the presence of a rapidly evolving multiprotein structure called the synaptonemal complex (SC). This study used whole-genome sequencing to investigate the fate of meiotic DSBs in D. melanogaster mutant females lacking functional SC, to assay for de novo copy-number variation (CNV) formation, and to examine the role of the SC in transposable element movement in flies. The data indicate that, in the absence of SC, copy-number variation still occurs and meiotic DSB repair by gene conversion occurs infrequently. Remarkably, an 856-kilobase de novo CNV was observed in two unrelated individuals of different genetic backgrounds and was identical to a CNV recovered in a previous wild-type study, suggesting that recurrent formation of large CNVs occurs in Drosophila. In addition, the rate of novel TE insertion was markedly higher than wild type in one of two SC mutants tested, suggesting that SC proteins may contribute to the regulation of TE movement and insertion in the genome. Overall, this study provides novel insight into the role that the SC plays in genome stability and provides clues as to why the sequence, but not structure, of SC proteins is rapidly evolving (Miller, 2019).

Nutrient-Driven O-GlcNAcylation Controls DNA Damage Repair Signaling and Stem/Progenitor Cell Homeostasis

Stem/progenitor cells exhibit high proliferation rates, elevated nutrient uptake, altered metabolic flux, and stress-induced genome instability. O-GlcNAcylation is an essential post-translational modification mediated by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which act in a nutrient- and stress-responsive manner. The precise role of O-GlcNAc in adult stem cells and the relationship between O-GlcNAc and the DNA damage response (DDR) is poorly understood. This study shows that hyper-O-GlcNacylation leads to elevated insulin signaling, hyperproliferation, and DDR activation that mimic the glucose- and oxidative-stress-induced response. A feedback mechanism was discovered involving key downstream effectors of DDR, ATM, ATR, and CHK1/2 that regulates OGT stability to promote O-GlcNAcylation and elevate DDR. This O-GlcNAc-dependent regulatory pathway is critical for maintaining gut homeostasis in Drosophila and the DDR in mouse embryonic stem cells (ESCs) and mouse embryonic fibroblasts (MEFs). These findings reveal a conserved mechanistic link among O-GlcNAc cycling, stem cell self-renewal, and DDR with profound implications for stem-cell-derived diseases including cancer (Na, 2020).

Mutations in the Drosophila rough deal gene affecting RZZ kinetochore function
The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10, and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the SAC by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. To explore the importance of the Rod_C domain in RZZ function in Drosophila, a series of point mutations was generated in a stretch of 200 residues within this domain, and their phenotypes are reported in this study. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores (Menant, 2020).

Loss of the spectraplakin gene Short stop induces a DNA damage response in Drosophila epithelia

Epithelia are an eminent tissue type and a common driver of tumorigenesis, requiring continual precision in cell division to maintain tissue structure and genome integrity. Mitotic defects often trigger apoptosis, impairing cell viability as a tradeoff for tumor suppression. Identifying conditions that lead to cell death and understanding the mechanisms behind this response are therefore of considerable importance. Here this study investigated how epithelia of the Drosophila wing disc respond to loss of Short stop (Shot), a cytoskeletal crosslinking spectraplakin protein that was previously found to control mitotic spindle assembly and chromosome dynamics. In contrast to other known spindle-regulating genes, Shot knockdown induces apoptosis in the absence of Jun kinase (JNK) activation, but instead leads to elevated levels of active p38 kinase. Shot loss leads to double-strand break (DSB) DNA damage, and the apoptotic response is exacerbated by concomitant loss of p53. DSB accumulation is increased by suppression of the spindle assembly checkpoint, suggesting this effect results from chromosome damage during error-prone mitoses. Consistent with DSB induction, DNA damage and stress response genes, Growth arrest and DNA damage (GADD45) and Apoptosis signal-regulating kinase 1 (Ask1), were found to be transcriptionally upregulated as part of the shot-induced apoptotic response. Finally, co-depletion of Shot and GADD45 induced significantly higher rates of chromosome segregation errors in cultured cells and suppressed shot-induced mitotic arrest. These results demonstrate that epithelia are capable of mounting molecularly distinct responses to loss of different spindle-associated genes and underscore the importance of proper cytoskeletal organization in tissue homeostasis (Dewey, 2020).

Involvement of DNA Repair Genes and System of Radiation-Induced Activation of Transposons in Formation of Transgenerational Effects

The study of the genetic basis of the manifestation of radiation-induced effects and their transgenerational inheritance makes it possible to identify the mechanisms of adaptation and possible effective strategies for the survival of organisms in response to chronic radioactive stress. One persistent hypothesis is that the activation of certain genes involved in cellular defense is a specific response of the cell to irradiation. There is also data indicating the important role of transposable elements in the formation of radiosensitivity/radioresistance of biological systems. This work studied the interaction of the systems of hobo transposon activity and DNA repair in the cell under conditions of chronic low-dose irradiation and its participation in the inheritance of radiation-induced transgenerational instability in Drosophila. The results showed a significant increase of sterility and locus-specific mutability, a decrease of survival, fertility and genome stability (an increase the frequency of dominant lethal mutations and DNA damage) in non-irradiated F(1)/F(2) offspring of irradiated parents with dysfunction of the mus304 gene which is responsible for excision and post-replicative recombination repair and repair of double-stranded DNA breaks. The combined action of dysfunction of the mus309 (Bloom syndrome helicase) gene and transpositional activity of hobo elements also led to the transgenerational effects of irradiation but only in the F(1) offspring. Dysfunction of the genes of other DNA repair systems (mus101 and mus210) showed no visible effects inherited from irradiated parents subjected to hobo transpositions. The mei-41 gene showed specificity in this type of interaction, which consists in its higher efficiency in sensing events induced by transpositional activity rather than irradiation (Yushkova, 2020).

A pathway for error-free non-homologous end joining of resected meiotic double-strand breaks. Nucleic Acids Res
Programmed DNA double-strand breaks (DSBs) made during meiosis are repaired by recombination with the homologous chromosome to generate, at selected sites, reciprocal crossovers that are critical for the proper separation of homologs in the first meiotic division. Backup repair processes can compensate when the normal meiotic recombination processes are non-functional. This paper describes a novel backup repair mechanism that occurs when the homologous chromosome is not available in Drosophila melanogaster meiosis. In the presence of a previously described mutation (Mcm5A7) that disrupts chromosome pairing, DSB repair is initiated by homologous recombination but is completed by non-homologous end joining (NHEJ). Remarkably, this process yields precise repair products. These results provide support for a recombination intermediate recently proposed in mouse meiosis, in which an oligonucleotide bound to the Spo11 protein that catalyzes DSB formation remains bound after resection. It is proposed that this oligonucleotide functions as a primer for fill-in synthesis to allow scarless repair by NHEJ. It is argued that this is a conserved repair mechanism that is likely to be invoked to overcome occasional challenges in normal meiosis (Hatkevich, 2021).


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Shindo, Y. and Amodeo, A. A. (2021). Excess histone H3 is a competitive Chk1 inhibitor that controls cell-cycle remodeling in the early Drosophila embryo. Curr Biol. PubMed ID: 33848457

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Zygotically transcribed genes

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