The Interactive Fly

Zygotically transcribed genes

DNA damage checkpoint, meiotic crossover, nondisjunction and repair

  • DNA damage-induced CHK2 activation compromises germline stem cell self-renewal and lineage differentiation
  • Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis
  • Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction
  • Annealing of complementary DNA sequences during double-strand break repair in Drosophila is mediated by the ortholog of SMARCAL1 DSA and end-joining pathways (Korda, 2017).
  • Substrate preference of Gen endonucleases highlights the importance of branched structures as DNA damage repair intermediates
  • Initiation of Drosophila chorion gene amplification requires Claspin and mus101, whereas Claspin, but not mus101, plays a major role during elongation
  • Mlh1 is required for female fertility in Drosophila melanogaster: An outcome of effects on meiotic crossing over, ovarian follicles and egg activation
  • Nuclear F-actin and myosins drive relocalization of heterochromatic breaks
  • Timely double-strand break repair and pathway choice in pericentromeric heterochromatin depend on the histone demethylase dKDM4A
  • Narya, a RING finger domain-containing protein, is required for meiotic DNA double-strand break formation and crossover maturation in Drosophila melanogaster
  • A robust transposon-endogenizing response from germline stem cells
  • Heterochromatic breaks move to the nuclear periphery to continue recombinational 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

    Bub1
    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

    Bub3
    mitotic checkpoint protein that serves as an essential protein required during normal mitotic progression to prevent premature sister
    chromatid separation, missegreation and aneuploidy

    grapes
    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

    nbs
    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

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

    p53
    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

    Xrp1
    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


    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).

    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 (Eyu, 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 (Eyu, 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 (Eyu, 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 (Eyu, 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 (Eyu, 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 (Eyu, 2015).


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    Zygotically transcribed genes

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