Bloom syndrome helicase: Biological Overview | References
Gene name - Bloom syndrome helicase
Synonyms - mus309, ku70
Cytological map position - 86E17-86E17
Function - enzyme, RecQ helicase
Keywords - repairing of 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
Symbol - Blm
FlyBase ID: FBgn0002906
Genetic map position - chr3R:11,736,810-11,741,783
NCBI lassification - Helicase superfamily c-terminal domain, RQC domain
Cellular location -
|Recent literature||Ertl, H. A., Russo, D. P., Srivastava, N., Brooks, J. T., Dao, T. N. and LaRocque, J. R. (2017). The Role of Blm Helicase in homologous recombination, gene conversion tract length, and recombination between diverged sequences in Drosophila. Genetics [Epub ahead of print]. PubMed ID: 28912341
DNA double-strand breaks (DSBs) are a particularly deleterious class of DNA damage that threatens genome integrity. DSBs are repaired by three pathways: non-homologous end joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA). Drosophila melanogaster Blm (DmBlm) is the ortholog of Saccharomyces cerevisiae SGS1 and human BLM, and has been shown to suppress crossovers in mitotic cells and repair mitotic DNA gaps via HR. To further elucidate the role of DmBlm in repair of a simple DSB, and in particular recombination mechanisms, the DR-white and DR-white.mu repair assays were used in multiple mutant allele backgrounds. DmBlm null and helicase-dead mutants both demonstrated a decrease in repair by noncrossover HR, and a concurrent increase in non-HR events, possibly including SSA, crossovers, deletions, and NHEJ, although detectable processing of the ends was not significantly impacted. Interestingly, gene conversion tract lengths of HR repair events were substantially shorter in DmBlm null but not helicase-dead mutants, compared to heterozygote controls. Using DR-white.mu, this study found that, in contrast to Sgs1, DmBlm is not required for suppression of recombination between diverged sequences. Taken together, these data suggest that DmBlm helicase function plays a role in HR, and the steps that contribute to determining gene conversion tract length are helicase-independent.
Studies in the yeast Saccharomyces cerevisiae have validated the major features of the double-strand break repair (DSBR) model as an accurate representation of the pathway through which meiotic crossovers are produced. This success has led to this model being invoked to explain double-strand break (DSB) repair in other contexts. However, most non-crossover recombinants generated during S. cerevisiae meiosis do not arise via a DSBR pathway. Furthermore, and it is becoming increasing clear that DSBR is a minor pathway for recombinational repair of DSBs that occur in mitotically proliferating cells; rather, the synthesis-dependent strand annealing (SDSA) model appears to describe mitotic DSB repair more accurately. Fundamental dissimilarities between meiotic and mitotic recombination are not unexpected, since meiotic recombination serves a very different purpose (accurate chromosome segregation, which requires crossovers) than mitotic recombination (repair of DNA damage, which typically generates non-crossovers) (Andersen, 2010).
Studies of the genetic requirements of non-crossover (NCO) repair have also provided important insights into the primary repair mechanisms. A key player in the process of actively promoting NCO repair and blocking CO-associated repair is the RecQ helicase Bloom (BLM) helicase. Cells lacking the BLM helicase have elevated spontaneous COs, primarily between sister chromatids, but also between homologous and heterologous chromosomes. Discussions of the anti-crossover functions of BLM are most often based on the hypothesis that the primary anti-crossover activity is in double Holliday junction (dHJ) dissolution (see Models for double-strand break repair). This hypothesis has been driven by a combination of genetic and biochemical studies (Andersen, 2010).
S. cerevisiae top3 mutants grow slowly, but the slow growth is suppressed by mutations in SGS1 (slow growth suppressor 1), which encodes the only RecQ helicase in this species. One interpretation of this finding is that Sgs1 produces an intermediate that is toxic in the absence of Top3 activity. The identity of the toxic intermediate is suggested by the in vitro dHJ dissolution activity of BLM and TOP3α. In vitro, BLM and TOP3α (together with accessory proteins) catalyze dHJ dissolution efficiently, with BLM migrating the HJs together so they can be decatenated by TOP3α. Thus, unresolved, branch-migrated dHJs may be the toxic intermediates in top3 mutants. However, direct evidence that dHJ dissolution occurs during mitotic DSB repair in vivo is lacking; indeed, it is now clear that dHJs are a minor intermediate in break repair in S. cerevisiae. The dissolvase activity of BLM - TOP3α may be important in preventing mitotic COs that arise from other types of spontaneous damage. For example, it has been hypothesized that repair of gaps that occur when replication is blocked on the lagging strand might involve formation of dHJ intermediates that do not originate with a DSB (Andersen, 2010).
Regardless of its roles in replication fork repair, Sgs1 and the Drosophila ortholog, DmBLM, do have important anti-crossover functions during DSB repair. Interestingly, mutants lacking DmBLM have severe defects in gap repair, suggesting that the primary role of DmBLM in preventing COs during break repair may be in promoting SDSA rather than in dissolving dHJs. It is possible that the genetic interaction between BLM and TOP3α reflects a heretofore uncharacterized requirement for TOP3α in synthesis-dependent strand annealing (SDSA). Perhaps TOP3α is required to relax supercoiling produced by D-loop production/migration. Such a requirement is unlikely to be revealed with the short substrates used in in vitro biochemical assays (Andersen, 2010).
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 shows 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, 2017).
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).
The Bloom syndrome helicase, BLM, has numerous functions that prevent mitotic crossovers. The unique features of Drosophila melanogaster were used to investigate origins and properties of mitotic crossovers that occur when BLM is absent. Induction of lesions that block replication forks increased crossover frequencies, consistent with functions for BLM in responding to fork blockage. In contrast, treatment with hydroxyurea, which stalls forks, did not elevate crossovers, even though mutants lacking BLM are sensitive to killing by this agent. To learn about sources of spontaneous recombination, mitotic crossovers were mapped in mutants lacking BLM. In the male germline, irradiation-induced crossovers were distributed randomly across the euchromatin, but spontaneous crossovers were nonrandom. It is suggested that regions of the genome with a high frequency of mitotic crossovers may be analogous to common fragile sites in the human genome. Interestingly, in the male germline there is a paucity of crossovers in the interval that spans the pericentric heterochromatin, but in the female germline this interval is more prone to crossing over. Finally, the assay system allowed recovery of pairs of reciprocal crossover chromosomes. Sequencing of these revealed the existence of gene conversion tracts and did not provide any evidence for mutations associated with crossovers. These findings provide important new insights into sources and structures of mitotic crossovers and functions of BLM helicase (LaFave, 2014).
Generation of meiotic crossovers in many eukaryotes requires the elimination of anti-crossover activities by using the Msh4-Msh5 heterodimer to block helicases. Msh4 and Msh5 have been lost from the flies Drosophila and Glossina, but a complex of minichromosome maintenance (MCM) proteins was identified that functionally replace Msh4-Msh5. REC, an ortholog of MCM8 that evolved under strong positive selection in flies, interacts with MEI-217 and MEI-218, which arose from a previously undescribed metazoan-specific MCM protein. Meiotic crossovers were reduced in Drosophila rec, mei-217, and mei-218 mutants; however, removal of the Bloom syndrome helicase (BLM) ortholog restored crossovers. Thus, MCMs were co-opted into a novel complex that replaced the meiotic pro-crossover function of Msh4-Msh5 in flies (Kohl, 2012).
The BLM DNA helicase plays a vital role in maintaining genome stability. Mutations in BLM cause Bloom syndrome, a rare disorder associated with cancer predisposition and premature aging. Humans and mice with blm mutations have increased frequencies of spontaneous mutagenesis, but the molecular basis of this increase is not well understood. In addition, the effect of aging on spontaneous mutagenesis in blm mutants has not been characterized. To address this, a lacZ reporter system was used in wild-type and several mutant strains of Drosophila melanogaster to analyze mechanisms of mutagenesis throughout their lifespan. The data show that Drosophila lacking BLM have an elevated frequency of spontaneous genome rearrangements that increases with age. Although in normal flies most genome rearrangements occur through DNA ligase 4-dependent classical end joining, most rearrangements that accumulate during aging in blm mutants do not require DNA ligase 4, suggesting the influence of an alternative end-joining mechanism. Adult blm mutants also display reduced lifespan and ligase 4-independent enhanced tumorigenesis in mitotically active tissues. These results suggest that Drosophila BLM suppresses error-prone alternative end-joining repair of DNA double-strand breaks that can result in genome instability and tumor formation during aging. In addition, since loss of BLM significantly affects lifespan and tumorigenesis, the data provide a link between error-prone end joining, genome rearrangements, and tumor formation in a model metazoan (Garcia, 2011).
DNA repair mechanisms in mitotically proliferating cells avoid generating crossovers, which can contribute to genome instability. Most models for the production of crossovers involve an intermediate with one or more four-stranded Holliday junctions (HJs), which are resolved into duplex molecules through cleavage by specialized endonucleases. In vitro studies have implicated three nuclear enzymes in HJ resolution: MUS81-EME1/Mms4, GEN1/Yen1, and SLX4-SLX1. The Bloom syndrome helicase, BLM, plays key roles in preventing mitotic crossover, either by blocking the formation of HJ intermediates or by removing HJs without cleavage. Saccharomyces cerevisiae mutants that lack Sgs1 (the BLM ortholog) and either Mus81-Mms4 or Slx4-Slx1 are inviable, but mutants that lack Sgs1 and Yen1 are viable. The current view is that Yen1 serves primarily as a backup to Mus81-Mms4. Previous studies with Drosophila melanogaster showed that, as in yeast, loss of both DmBLM and MUS81 or MUS312 (the ortholog of SLX4) is lethal. This study has now recovered and analyzed mutations in Drosophila Gen. As in yeast, there is some redundancy between Gen and mus81; however, in contrast to the case in yeast, GEN plays a more predominant role in responding to DNA damage than MUS81-MMS4. Furthermore, loss of DmBLM and GEN leads to lethality early in development. A comparison is presented of phenotypes occurring in double mutants that lack DmBLM and either MUS81, GEN, or MUS312, including chromosome instability and deficiencies in cell proliferation. These studies of synthetic lethality provide insights into the multiple functions of DmBLM and how various endonucleases may function when DmBLM is absent (Andersen, 2011).
Bloom Syndrome, a rare human disorder characterized by genomic instability and predisposition to cancer, is caused by mutation of BLM, which encodes a RecQ-family DNA helicase. The Drosophila melanogaster ortholog of BLM, DmBlm, is encoded by mus309. Mutations in mus309 cause hypersensitivity to DNA-damaging agents, female sterility, and defects in repairing double-strand breaks (DSBs). To better understand these phenotypes, novel mus309 alleles were isolated. Mutations that delete the N terminus of DmBlm, but not the helicase domain, have DSB repair defects as severe as those caused by null mutations. Female sterility is due to a requirement for DmBlm in early embryonic cell cycles; embryos lacking maternally derived DmBlm have anaphase bridges and other mitotic defects. These defects were less severe for the N-terminal deletion alleles, so one of these mutations was used to assay meiotic recombination. Crossovers were decreased to about half the normal rate, and the remaining crossovers were evenly distributed along the chromosome. Spontaneous mitotic crossovers are increased by several orders of magnitude in mus309 mutants. These results demonstrate that DmBlm functions in multiple cellular contexts to promote genome stability (McVey, 2007).
RecQ helicases are critical for maintaining genomic integrity. This study shows that three RecQ members (WRN, deficient in the Werner syndrome; BLM, deficient in the Bloom syndrome; and Drosophila melanogaster RecQ5b (dmRecQ5b)) possess a novel strand pairing activity. Furthermore, each of these enzymes combines this strand pairing activity with its inherent DNA unwinding capability to perform coordinated strand exchange. In this regard, WRN and BLM are considerably more efficient than dmRecQ5b, apparently because dmRecQ5b lacks conserved sequences C-terminal to the helicase domain that contribute to DNA binding, strand pairing, and strand exchange. Based on these findings, it is postulated that certain RecQ helicases are structurally designed to accomplish strand exchange on complex replication and recombination intermediates. This is highly consistent with proposed roles for RecQ members in DNA metabolism and the illegitimate recombination and cancer-prone phenotypes associated with RecQ defects (Machwe, 2005).
P transposable elements in Drosophila are mobilized via a cut-and-paste mechanism. The broken DNA ends generated during transposition can be repaired via the homology-directed synthesis-dependent strand annealing or by nonhomologous end joining (NHEJ). Genetic studies have demonstrated an interaction between the gene (mus309, for mutagen-sensitive) encoding the Drosophila Bloom's syndrome helicase homolog (DmBLM) and the Ku70 gene, which is involved in NHEJ. This study used RNA interference (RNAi) to knock down expression of DmBLM and one or both of the Drosophila Ku subunits, DmKu70 or DmKu80. The results show that upon reduction of DmKu, an increase in small deletions (1-49 bp) and large deletions (>/=50 bp) flanking the site of P element-induced breaks is observed, and a reduction in large deletions at these sites is found upon reduction of DmBLM. Moreover, double RNAi of DmKu and DmBLM results in an increase in small deletions characteristic of the DmKu RNAi and also partially suppresses the reduction in repair efficiency observed with DmKu RNAi. These results suggest that there are DNA double-strand break recognition and/or processing events involving DmKu and DmBLM that, when eliminated by RNAi, lead to deletions. Finally, these results raise the possibility that, unlike the situation in mammals, where BLM appears to function exclusively in the homologous repair pathway, in Drosophila, DmBLM may be directly involved in, or at least influence the double-strand break recognition that leads to the NHEJ repair pathway (Min, 2004).
Several eukaryotic homologs of the Escherichia coli RecQ DNA helicase have been found. These include the human BLM gene, whose mutation results in Bloom syndrome, and the human WRN gene, whose mutation leads to Werner syndrome resembling premature aging. A Drosophila melanogaster homolog of the RECQ helicase family, Dmblm (Drosophila melanogaster Bloom), which encodes a putative 1487-amino-acid protein, was cloned in this study. Phylogenetic and dot plot analyses for the RECQ family, including 10 eukaryotic and 3 prokaryotic genes, indicate Dmblm is most closely related to the Homo sapiens BLM gene, suggesting functional similarity. Dmblm cDNA partially rescued the sensitivity to methyl methanesulfonate of Saccharomyces cerevisiae sgs1 mutant, demonstrating the presence of a functional similarity between Dmblm and SGS1. This analyses identified four possible subfamilies in the RECQ family: (1) the BLM subgroup (H. sapiens Bloom, D. melanogaster Dmblm, and Caenorhabditis elegans T04A11.6); (2) the yeast RECQ subgroup (S. cerevisiae SGS1 and Schizosaccharomyces pombe rqh1/rad12); (3) the RECQL/Q1 subgroup (H. sapiens RECQL/Q1 and C. elegans K02F3.1); and (4) the WRN subgroup (H. sapiens Werner and C. elegans F18C5.2). This result may indicate that metazoans hold at least three RECQ genes, each of which may have a different function, and that multiple RECQ genes diverged with the generation of multicellular organisms. It is proposed that invertebrates such as nematodes and insects are useful as model systems of human genetic diseases (Kusano, 1999).
The recruitment of FANCM, a conserved DNA translocase and key component of several DNA repair protein complexes, to replication forks stalled by DNA interstrand crosslinks (ICLs) is a step upstream of the Fanconi anemia (FA) repair and replication traverse pathways of ICLs. However, detection of the FANCM recruitment has been technically challenging so that its mechanism remains exclusive. This study successfully observed recruitment of FANCM at stalled forks using a newly developed protocol. The FANCM recruitment depends upon its intrinsic DNA translocase activity, and its DNA-binding partner FAAP24. Moreover, it is dependent on the replication checkpoint kinase, ATR; but is independent of the FA core and FANCD2-FANCI complexes, two essential components of the FA pathway, indicating that the FANCM recruitment occurs downstream of ATR but upstream of the FA pathway. Interestingly, the recruitment of FANCM requires its direct interaction with Bloom syndrome complex composed of BLM helicase, Topoisomerase 3α, RMI1 and RMI2; as well as the helicase activity of BLM. It was further shown that the FANCM-BLM complex interaction is critical for replication stress-induced FANCM hyperphosphorylation, for normal activation of the FA pathway in response to ICLs, and for efficient traverse of ICLs by the replication machinery. Epistasis studies demonstrate that FANCM and BLM work in the same pathway to promote replication traverse of ICLs. It is concluded that FANCM and BLM complex work together at stalled forks to promote both FA repair and replication traverse pathways of ICLs (Ling, 2016).
Meiotic recombination is essential for the repair of programmed double strand breaks (DSBs) to generate crossovers (COs) during meiosis. The efficient processing of meiotic recombination intermediates not only needs various resolvases but also requires proper meiotic chromosome structure. The Smc5/6 complex belongs to the structural maintenance of chromosome (SMC) family and is closely related to cohesin and condensin. Although the Smc5/6 complex has been implicated in the processing of recombination intermediates during meiosis, it is not known how Smc5/6 controls meiotic DSB repair. Using Caenorhabditis elegans this study shows that the SMC-5/6 complex acts synergistically with HIM-6, an ortholog of the human Bloom syndrome helicase (BLM) during meiotic recombination. The concerted action of the SMC-5/6 complex and HIM-6 is important for processing recombination intermediates, CO regulation and bivalent maturation. Careful examination of meiotic chromosomal morphology reveals an accumulation of inter-chromosomal bridges in smc-5; him-6 double mutants, leading to compromised chromosome segregation during meiotic cell divisions. Interestingly, t the lethality of smc-5; him-6 can be rescued by loss of the conserved BRCA1 ortholog BRC-1. Furthermore, the combined deletion of smc-5 and him-6 leads to an irregular distribution of condensin and to chromosome decondensation defects reminiscent of condensin depletion. Lethality conferred by condensin depletion can also be rescued by BRC-1 depletion. These results suggest that SMC-5/6 and HIM-6 can synergistically regulate recombination intermediate metabolism and suppress ectopic recombination by controlling chromosome architecture during meiosis (Hong, 2016).
Based on its in vitro unwinding activity on G-quadruplex (G4) DNA, the Bloom syndrome-associated helicase BLM is proposed to participate in telomere replication by aiding fork progression through G-rich telomeric DNA. Single molecule analysis of replicated DNA (SMARD) was used to determine the contribution of BLM helicase to telomere replication. In BLM-deficient cells, replication forks initiating from origins within the telomere, which copy the G-rich strand by leading strand synthesis, moved slower through the telomere compared with the adjacent subtelomere. Fork progression through the telomere was further slowed in the presence of a G4 stabilizer. Using a G4-specific antibody, it was found that deficiency of BLM, or another G4-unwinding helicase, the Werner syndrome-associated helicase WRN, resulted in increased G4 structures in cells. Importantly, deficiency of either helicase led to greater increases in G4 DNA detected in the telomere compared with G4 seen genome-wide. Collectively, these findings are consistent with BLM helicase facilitating telomere replication by resolving G4 structures formed during copying of the G-rich strand by leading strand synthesis (Drosopoulos, 2015).
Bloom's syndrome helicase (BLM) is a member of the RecQ family of DNA helicases, which play key roles in the maintenance of genome integrity in all organism groups. This study describes crystal structures of the BLM helicase domain in complex with DNA and with an antibody fragment, as well as SAXS and domain association studies in solution. An unexpected nucleotide-dependent interaction is shown of the core helicase domain with the conserved, poorly characterized HRDC domain. The BLM-DNA complex shows an unusual base-flipping mechanism with unique positioning of the DNA duplex relative to the helicase core domains. Comparison with other crystal structures of RecQ helicases permits the definition of structural transitions underlying ATP-driven helicase action, and the identification of a nucleotide-regulated tunnel that may play a role in interactions with complex DNA substrates (Newman, 2015).
Bloom syndrome caused by inactivation of the Bloom DNA helicase (Blm) is characterized by increases in the level of sister chromatid exchange, homologous recombination (HR) associated with cross-over. It is therefore believed that Blm works as an anti-recombinase. Meanwhile, in Drosophila, DmBlm is required specifically to promote the synthesis-dependent strand anneal (SDSA), a type of HR not associating with cross-over. However, conservation of Blm function in SDSA through higher eukaryotes has been a matter of debate. This study demonstrates the function of Blm in SDSA type HR in chicken DT40 B lymphocyte line, where Ig gene conversion diversifies the immunoglobulin V gene through intragenic HR between diverged homologous segments. This reaction is initiated by the activation-induced cytidine deaminase enzyme-mediated uracil formation at the V gene, which in turn converts into abasic site, presumably leading to a single strand gap. Ig gene conversion frequency was drastically reduced in BLM(-/-) cells. In addition, BLM(-/-) cells used limited donor segments harboring higher identity compared with other segments in Ig gene conversion event, suggesting that Blm can promote HR between diverged sequences. To further understand the role of Blm in HR between diverged homologous sequences, the frequency was measured of gene targeting induced by an I-SceI-endonuclease-mediated double-strand break. BLM(-/-) cells showed a severer defect in the gene targeting frequency as the number of heterologous sequences increased at the double-strand break site. Conversely, the overexpression of Blm, even an ATPase-defective mutant, strongly stimulated gene targeting. In summary, Blm promotes HR between diverged sequences through a novel ATPase-independent mechanism (Kikuchi, 2009).
Search PubMed for articles about Drosophila Bloom syndrome helicase
Andersen, S. L. and Sekelsky, J. (2010). Meiotic versus mitotic recombination: two different routes for double-strand break repair: the different functions of meiotic versus mitotic DSB repair are reflected in different pathway usage and different outcomes. Bioessays 32(12): 1058-1066. PubMed ID: 20967781
Andersen, S. L., Kuo, H. K., Savukoski, D., Brodsky, M. H. and Sekelsky, J. (2011). Three structure-selective endonucleases are essential in the absence of BLM helicase in Drosophila. PLoS Genet 7(10): e1002315. PubMed ID: 22022278
Drosopoulos, W. C., Kosiyatrakul, S. T. and Schildkraut, C. L. (2015). BLM helicase facilitates telomere replication during leading strand synthesis of telomeres. J Cell Biol 210(2): 191-208. PubMed ID: 26195664
Garcia, A. M., Salomon, R. N., Witsell, A., Liepkalns, J., Calder, R. B., Lee, M., Lundell, M., Vijg, J. and McVey, M. (2011). Loss of the Bloom syndrome helicase increases DNA ligase 4-independent genome rearrangements and tumorigenesis in aging Drosophila. Genome Biol 12(12): R121. PubMed ID: 22183041
Hatkevich, T., Kohl, K. P., McMahan, S., Hartmann, M. A., Williams, A. M. and Sekelsky, J. (2016). Bloom syndrome helicase promotes meiotic crossover patterning and homolog disjunction. Curr Biol [Epub ahead of print]. PubMed ID: 27989672
Hong, Y., Sonneville, R., Agostinho, A., Meier, B., Wang, B., Blow, J. J. and Gartner, A. (2016). The SMC-5/6 Complex and the HIM-6 (BLM) Helicase Synergistically Promote Meiotic Recombination Intermediate Processing and Chromosome Maturation during Caenorhabditis elegans Meiosis. PLoS Genet 12(3): e1005872. PubMed ID: 27010650
Kikuchi, K., Abdel-Aziz, H. I., Taniguchi, Y., Yamazoe, M., Takeda, S. and Hirota, K. (2009). Bloom DNA helicase facilitates homologous recombination between diverged homologous sequences. J Biol Chem 284(39): 26360-26367. PubMed ID: 19661064
Kohl, K. P., Jones, C. D. and Sekelsky, J. (2012). Evolution of an MCM complex in flies that promotes meiotic crossovers by blocking BLM helicase. Science 338(6112): 1363-1365. PubMed ID: 23224558
Kusano, K., Berres, M. E. and Engels, W. R. (1999). Evolution of the RECQ family of helicases: A drosophila homolog, Dmblm, is similar to the human bloom syndrome gene. Genetics 151(3): 1027-1039. PubMed ID: 10049920
LaFave, M. C., Andersen, S. L., Stoffregen, E. P., Holsclaw, J. K., Kohl, K. P., Overton, L. J. and Sekelsky, J. (2014). Sources and structures of mitotic crossovers that arise when BLM helicase is absent in Drosophila. Genetics 196(1): 107-118. PubMed ID: 24172129
Ling, C., Huang, J., Yan, Z., Li, Y., Ohzeki, M., Ishiai, M., Xu, D., Takata, M., Seidman, M. and Wang, W. (2016). Bloom syndrome complex promotes FANCM recruitment to stalled replication forks and facilitates both repair and traverse of DNA interstrand crosslinks. Cell Discov 2: 16047. PubMed ID: 28058110
Machwe, A., Xiao, L., Groden, J., Matson, S. W. and Orren, D. K. (2005). RecQ family members combine strand pairing and unwinding activities to catalyze strand exchange. J Biol Chem 280(24): 23397-23407. PubMed ID: 15845538
McVey, M., Andersen, S. L., Broze, Y. and Sekelsky, J. (2007). Multiple functions of Drosophila BLM helicase in maintenance of genome stability. Genetics 176(4): 1979-1992. PubMed ID: 17507683
Min, B., Weinert, B. T. and Rio, D. C. (2004). Interplay between Drosophila Bloom's syndrome helicase and Ku autoantigen during nonhomologous end joining repair of P element-induced DNA breaks. Proc Natl Acad Sci U S A 101(24): 8906-8911. PubMed ID: 15184650
Newman, J. A., Savitsky, P., Allerston, C. K., Bizard, A. H., Ozer, O., Sarlos, K., Liu, Y., Pardon, E., Steyaert, J., Hickson, I. D. and Gileadi, O. (2015). Crystal structure of the Bloom's syndrome helicase indicates a role for the HRDC domain in conformational changes. Nucleic Acids Res 43(10): 5221-5235. PubMed ID: 25901030
date revised: 15 January 2017
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