Division of Labor by the HELQ, BLM, and FANCM Helicases during Homologous Recombination Repair in Drosophila melanogaster

Repair of DNA double-strand breaks by homologous recombination (HR) requires a carefully orchestrated sequence of events involving many proteins. One type of HR, synthesis-dependent strand annealing (SDSA), proceeds via the formation of a displacement loop (D-loop) when RAD51-coated single-stranded DNA invades a homologous template. The 3′ end of the single-stranded DNA is extended by DNA synthesis. In SDSA, the D-loop is then disassembled prior to strand annealing. While many helicases can unwind D-loops in vitro, how their action is choreographed in vivo remains to be determined. To clarify the roles of various DNA helicases during SDSA, we used a double-strand gap repair assay to study the outcomes of homologous recombination repair in Drosophila melanogaster lacking the BLM, HELQ, and FANCM helicases. We found that the absence of any of these three helicases impairs gap repair. In addition, flies lacking both BLM and HELQ or HELQ and FANCM had more severe SDSA defects than the corresponding single mutants. In the absence of BLM, a large percentage of repair events were accompanied by flanking deletions. Strikingly, these deletions were mostly abolished in the blm helq and blm fancm double mutants. Our results suggest that the BLM, HELQ, and FANCM helicases play distinct roles during SDSA, with HELQ and FANCM acting early to promote the formation of recombination intermediates that are then processed by BLM to prevent repair by deletion-prone mechanisms.


Introduction
Homologous recombination (HR) is a high-fidelity mechanism of DNA double-strand break repair. In eukaryotes, the initial steps of HR involve 3 to 5 resection of DNA ends by an orchestrated collection of nucleases and helicases. The RAD51 protein is loaded onto the resultant single-stranded DNA (ssDNA) and the nucleoprotein filament promotes a search for homologous repair templates, such as a sister chromatid, homologous chromosome, or ectopic homologous sequence. RAD51-coated ssDNA invades the template and forms a displacement loop (D-loop) intermediate, which is then extended by DNA polymerase delta [1], with possible contributions from translesion polymerases eta and zeta [2][3][4][5][6][7]. The D-loop enlarges until it is about 100-200 nt in size, at which time it converts to a migrating D-loop as synthesis proceeds [6]. In the synthesis-dependent strand annealing (SDSA) model of HR, the D-loop is unwound by one or more DNA helicases, and the nascent DNA anneals to complementary sequences on the other side of the break [8]. Repair concludes with fill-in synthesis and ligation. Importantly, SDSA proceeds without the formation of Holliday junctions, thereby preventing mitotic crossovers that can lead to the loss of heterozygosity (reviewed in [9][10][11]). strand break repair in mitotic cells, we created a deletion mutation of HELQ by imprecise excision of P(SUPor-P)mus301 KG09098 . The helq 288A mutation removes more than 2 kilobases of coding sequence, including the entire helicase domain ( Figure 1A). We confirmed that helq mutants are sensitive to nitrogen mustard at a level comparable to homologous recombination-deficient spn-A mutants lacking the RAD51 recombinase ( Figure 1B). We then tested the helq mutant for sensitivity to topotecan, a topoisomerase I inhibitor that induces one-ended DNA double-strand breaks in replicating cells. Flies lacking HELQ were hypersensitive to topotecan, with relative survival slightly less than spn-A mutants ( Figure 1C).

HELQ Is Required for Homologous Recombination Repair of Double-Strand Breaks
Drosophila HELQ (encoded by the spn-C/mus301 gene) is required for survival following exposure to methyl methanesulfonate and nitrogen mustard and for the repair of meiotic double-strand breaks [34,39]. To determine whether HELQ is required for doublestrand break repair in mitotic cells, we created a deletion mutation of HELQ by imprecise excision of P(SUPor-P)mus301 KG09098 . The helq 288A mutation removes more than 2 kilobases of coding sequence, including the entire helicase domain ( Figure 1A). We confirmed that helq mutants are sensitive to nitrogen mustard at a level comparable to homologous recombination-deficient spn-A mutants lacking the RAD51 recombinase ( Figure 1B). We then tested the helq mutant for sensitivity to topotecan, a topoisomerase I inhibitor that induces one-ended DNA double-strand breaks in replicating cells. Flies lacking HELQ were hypersensitive to topotecan, with relative survival slightly less than spn-A mutants ( Figure 1C). To further investigate the role of HELQ in HR repair, we tested helq mutants using a well-characterized gap repair assay [40]. In this assay, a 14 kilobase P element carrying the white gene is inserted on the X chromosome ( Figure 2A). The 5′ and 3′ regions of white are interrupted by a copia retrotransposon, which decreases white expression so that female progeny with two copies of P{w a } have apricot-colored eyes and females with one copy have yellow eyes. Expression of transposase causes infrequent excision of the P To further investigate the role of HELQ in HR repair, we tested helq mutants using a well-characterized gap repair assay [40]. In this assay, a 14 kilobase P element carrying the white gene is inserted on the X chromosome ( Figure 2A). The 5 and 3 regions of white are interrupted by a copia retrotransposon, which decreases white expression so that female progeny with two copies of P{w a } have apricot-colored eyes and females with one copy have yellow eyes. Expression of transposase causes infrequent excision of the P element, creating a 14 kilobase double-stranded gap relative to the uncut sister chromatid. Excision of P{w a } and repair can occur in both somatic and germline tissues. copia and annealing at the LTRs removes most of copia, creating a product with increased white expression (SDSA + LTR annealing; Figure 2C). For unknown reasons, synthesis sometimes aborts, and repair concludes through a DNA ligase 4-independent alternative end-joining process [41] (aborted SDSA; Figure 2C). Red-eyed F1 female progeny inherit SDSA + LTR annealing products that involve at least 4 kilobases of synthesis on each side, while yellow-eyed female progeny inherit products that result from aborted SDSA and alternative end joining. The P(w a ) transposon, located on the X chromosome, contains a complete copy of the white gene interrupted by the copia retrotransposon. The presence of copia reduces the expression of white, resulting in yellow-eyed females if present in one copy and apricot-eyed females if present in two copies. (B) P-element excision is stimulated in males in the presence of P transposase, creating a 14 kilobase double-stranded gap. Repair events occurring in the male pre-meiotic germline (P{w a }*) are recovered in F1 females with an intact copy of P{w a }. The amount of repair synthesis that occurred during SDSA is quantified by PCR using genomic DNA from white-eyed F2 male progeny. (C) HR repair involving two-ended strand invasion into an uncut The P{w a } transposon, located on the X chromosome, contains a complete copy of the white gene interrupted by the copia retrotransposon. The presence of copia reduces the expression of white, resulting in yellow-eyed females if present in one copy and apricot-eyed females if present in two copies. (B) P-element excision is stimulated in males in the presence of P transposase, creating a 14 kilobase double-stranded gap. Repair events occurring in the male pre-meiotic germline (P{w a }*) are recovered in F 1 females with an intact copy of P{w a }. The amount of repair synthesis that occurred during SDSA is quantified by PCR using genomic DNA from white-eyed F 2 male progeny. (C) HR repair involving two-ended strand invasion into an uncut sister chromatid, synthesis, and annealing at the copia LTRs deletes most of copia, restoring white expression and resulting in red-eyed female progeny (top). Aborted HR repair in which end-joining occurs prior to synthesis of white results in yellow-eyed female progeny (middle). Repair involving deletions into flanking sd sequences results in scalloped-winged females and/or lethality in F 2 males (bottom).
Repair pathway choice is easily quantified by mating males undergoing excision and repair in their germlines to females possessing two copies of the P{w a } element, then scoring eye color in the F 1 female progeny ( Figure 2B). Strand invasion can occur on both sides of the gap, resulting in two D-loops that migrate toward each other as repair synthesis proceeds. Bidirectional synthesis past the 276 base-pair long terminal repeats (LTRs) of copia and annealing at the LTRs removes most of copia, creating a product with increased white expression (SDSA + LTR annealing; Figure 2C). For unknown reasons, synthesis sometimes aborts, and repair concludes through a DNA ligase 4-independent alternative end-joining process [41] (aborted SDSA; Figure 2C). Red-eyed F 1 female progeny inherit SDSA + LTR annealing products that involve at least 4 kilobases of synthesis on each side, while yellow-eyed female progeny inherit products that result from aborted SDSA and alternative end joining.
Males lacking HELQ showed a significant decrease in SDSA + LTR annealing repair events compared to the wild type, with a corresponding increase in end-joining repair following aborted HR ( Figure 3A,B). This, combined with the topotecan sensitivity of helq mutants, suggests that HELQ plays an important mitotic role in the HR repair of double-strand breaks. To determine the extent of repair synthesis that occurred during incomplete SDSA in wild type and helq mutants, we performed PCR on genomic DNA isolated from F 2 white-eyed males. Interestingly, helq mutant males had decreased repair synthesis at all distances measured ( Figure 3C), suggesting that HELQ is important for processive synthesis during gap repair.
Genes 2022, 13, x FOR PEER REVIEW 6 of 14 sister chromatid, synthesis, and annealing at the copia LTRs deletes most of copia, restoring white expression and resulting in red-eyed female progeny (top). Aborted HR repair in which end-joining occurs prior to synthesis of white results in yellow-eyed female progeny (middle). Repair involving deletions into flanking sd sequences results in scalloped-winged females and/or lethality in F2 males (bottom).
Males lacking HELQ showed a significant decrease in SDSA + LTR annealing repair events compared to the wild type, with a corresponding increase in end-joining repair following aborted HR ( Figure 3A,B). This, combined with the topotecan sensitivity of helq mutants, suggests that HELQ plays an important mitotic role in the HR repair of doublestrand breaks. To determine the extent of repair synthesis that occurred during incomplete SDSA in wild type and helq mutants, we performed PCR on genomic DNA isolated from F2 white-eyed males. Interestingly, helq mutant males had decreased repair synthesis at all distances measured ( Figure 3C), suggesting that HELQ is important for processive synthesis during gap repair.

HELQ Plays a Role in SDSA Distinct from That of the BLM Helicase
The decreased use of SDSA + LTR annealing repair and shorter repair synthesis tracts observed for helq mutants mimic the phenotypes previously observed in flies lacking BLM [23,24]. Thus, to determine if BLM and HELQ perform different functions during HR, we tested blm single mutants and helq blm double mutants using the P{w a } assay. As previously reported, the blm mutants had a decreased percentage of SDSA + LTR annealing events and increased aborted SDSA repair compared to wild-type males ( Figure 3A,B). Aborted SDSA repair events recovered from blm males also had shorter repair synthesis tracts than the wild type ( Figure 3C). In the helq blm double mutants, we observed a Wild type (n = 74), helq 288A (n = 67), blm N1 (n = 71), helq 288A blm N1 (n = 70). Statistical comparisons between all mutants can be found in Supplemental Tables S1-S3.

HELQ Plays a Role in SDSA Distinct from That of the BLM Helicase
The decreased use of SDSA + LTR annealing repair and shorter repair synthesis tracts observed for helq mutants mimic the phenotypes previously observed in flies lacking BLM [23,24]. Thus, to determine if BLM and HELQ perform different functions during HR, we tested blm single mutants and helq blm double mutants using the P{w a } assay. As previously reported, the blm mutants had a decreased percentage of SDSA + LTR annealing events and increased aborted SDSA repair compared to wild-type males ( Figure 3A,B). Aborted SDSA repair events recovered from blm males also had shorter repair synthesis tracts than the wild type ( Figure 3C). In the helq blm double mutants, we observed a significant decrease in SDSA + LTR annealing repair compared to the blm but not the helq mutants ( Figure 3A). The levels of aborted SDSA repair were similar between both single mutants and the helq blm double mutant ( Figure 3B). However, the repair synthesis tracts of the double mutants were significantly shorter than those of the single mutants at all but the longest distance ( Figure 3C), indicating that HELQ and BLM either act at different stages of SDSA or are individually limiting for repair synthesis.
In blm mutants, aborted SDSA repair events are frequently accompanied by flanking deletions that impact the expression of the essential sd gene, resulting in a scalloped-wing phenotype and/or male lethality in the F 2 generation [25] ( Figure 2C). To analyze deletion frequency in various genetic backgrounds, we quantified the frequency of F 1 scallopedwinged females and F 2 male lethal deletions. In contrast to the blm mutants, we observed very few lethal deletions in the helq mutants (Table 1). Interestingly, there was partial suppression of both scalloped females and male-lethal deletions in the double mutant. Table 1. Loss of HELQ partially suppresses the number of lethal flanking deletions that occur in the absence of BLM. The P{w a } construct is inserted in an intron of scalloped (sd), an X chromosome gene required for viability. F 1 females inheriting an aborted SDSA repair event with a flanking deletion sometimes have malformed wings (left column), while males inheriting large deletions into the sd coding sequence do not survive (right column). Parenthetical numbers indicate the total number of events analyzed: left, total number of yellow-eyed F 1 females counted; right, total number of independent yellow-eyed F 1 females from which F 2 males were counted. a: significantly increased from WT (P < 0.0001); b: significantly different from both single mutants (P < 0.0001); Fisher's exact test.

Genotype % Scalloped-Winged Females (F 1 ) % Male-Lethal Deletions (F 2 )
Wild type 0.0% (84) 0.0% (44 Smaller flanking deletions that extend into the scalloped intronic sequence but do not affect scalloped expression may be non-lethal and remain undetected by our deletion analysis in Table 1. A negative PCR result for the DNA synthesis analysis at 5 bp on the right end of P{w a } could indicate either a lack of synthesis from that end or a non-lethal flanking deletion. To discern between these two possibilities, we analyzed these events using primers that anneal to the scalloped intronic sequence, either to the left or the right of the insertion site. Similar to the large deletion frequency, small deletions were common in blm mutants but not in helq mutants (Table 2). In the helq blm double mutant, we again saw suppression of the blm phenotype. Together with the large deletion analysis, these data suggest that HELQ may promote the formation of an SDSA intermediate that requires BLM for its resolution. In the absence of BLM, deletion-prone repair predominates. Table 2. Increased non-lethal flanking deletions in blm mutants but not helq or helq blm mutants. Aberrant repair events that were negative for synthesis from the left or right end of P{w a } were analyzed for small flanking deletions into scalloped intron sequences. a: significantly increased from helq mutants (P < 0.05); b: significantly decreased from blm mutants (P < 0.05); Fisher's exact test.

FANCM Helicase Has a Minor Role in SDSA
The FANCM protein has also been shown to promote SDSA repair, although fancm mutant defects in the P{w a } assay are less severe than those in blm mutants [27]. To determine if the roles of FANCM and BLM in SDSA overlap, we compared fancm and blm single and double mutants using the P{w a } gap repair assay. The blm mutations used were blm N1 , blm N2 , and blm D2 , all of which cause similar defects in SDSA repair [23]. As previously reported, fancm mutants had fewer SDSA + LTR repair events compared to the wild type, although the decrease was not as large as with the blm mutants [27] (Figure 4A). blm mutants also had a greater percentage of aborted SDSA repair products compared to fancm mutants ( Figure 4B), and the amount of DNA synthesis from the right end of the gap was less in blm mutants than in fancm mutants ( Figure 4C).
The FANCM protein has also been shown to promote SDSA repair, although fancm mutant defects in the P{w a } assay are less severe than those in blm mutants [27]. To determine if the roles of FANCM and BLM in SDSA overlap, we compared fancm and blm single and double mutants using the P{w a } gap repair assay. The blm mutations used were blm N1 , blm N2 , and blm D2 , all of which cause similar defects in SDSA repair [23]. As previously reported, fancm mutants had fewer SDSA + LTR repair events compared to the wild type, although the decrease was not as large as with the blm mutants [27] ( Figure  4A). blm mutants also had a greater percentage of aborted SDSA repair products compared to fancm mutants ( Figure 4B), and the amount of DNA synthesis from the right end of the gap was less in blm mutants than in fancm mutants ( Figure 4C).
Interestingly, the percentages of SDSA + LTR annealing products and aborted SDSA products were similar in the blm and blm fancm mutants ( Figure 4A,B). In addition, the amount of repair synthesis at all distances measured in the double mutant was not statistically different from that in the blm mutant ( Figure 4C and Supplemental Table S3). To further probe the differences between the fancm and blm mutants, we quantified the percentage of deletion events in each of the single mutants and compared them to the blm fancm double mutant. In contrast to the blm mutant phenotype, the loss of FANCM did not significantly increase the frequency of repair events with large deletions (Table 3). Intriguingly, mutation of FANCM partially suppressed the deletion-prone repair of the blm mutants, similar to what we observed with the helq blm double mutants.  Interestingly, the percentages of SDSA + LTR annealing products and aborted SDSA products were similar in the blm and blm fancm mutants ( Figure 4A,B). In addition, the amount of repair synthesis at all distances measured in the double mutant was not statistically different from that in the blm mutant ( Figure 4C and Supplemental Table S3). To further probe the differences between the fancm and blm mutants, we quantified the percentage of deletion events in each of the single mutants and compared them to the blm fancm double mutant. In contrast to the blm mutant phenotype, the loss of FANCM did not significantly increase the frequency of repair events with large deletions (Table 3). Intriguingly, mutation of FANCM partially suppressed the deletion-prone repair of the blm mutants, similar to what we observed with the helq blm double mutants. Table 3. Loss of FANCM partially suppresses the number of lethal flanking deletions that occur in the absence of BLM. F 1 females with adeletion affecting the scalloped gene (left column) or causing lethality in males inheriting the deletion (right column). Parenthetical numbers indicate the total number of events analyzed: left, total number of yellow-eyed F 1 females counted; right, total number of independent yellow-eyed F 1 females from which F 2 males were counted. The wild-type data are the same as in Table 1. a: significantly different from the wild type and fancm mutant (P < 0.00001); b: significantly different from the wild type and both single mutants (P < 0.05); Fisher's exact test.

Loss of Both HELQ and FANCM Has Additive Effects on Repair Synthesis during SDSA
The data from the gap repair assay suggest that both HELQ and FANCM play roles distinct from the BLM helicase in SDSA repair. To determine whether HELQ and FANCM may also act independently from each other during HR, we constructed helq fancm double mutants and tested them in the P{wa} assay.
While loss of either helicase resulted in a significant decrease in red-eyed progeny compared to the wild type, mutation of HELQ caused a more severe SDSA defect compared to mutation of FANCM ( Figure 5A). There was no additional significant decrease in redeyed progeny in the helq fancm double mutant compared to the helq mutant, nor was the frequency of aborted SDSA further elevated in the helq fancm double mutant compared to the helq mutant ( Figure 5B). However, when repair synthesis in the aborted SDSA events was quantified by PCR, we observed significantly shorter repair synthesis tracts in the helq fancm double mutant compared to either single mutant ( Figure 5C and Supplemental Table S3). This suggests that HELQ and FANCM may have non-overlapping roles during SDSA. Alternatively, each protein may be limiting for a process that promotes repair synthesis.
Genes 2022, 13, x FOR PEER REVIEW 9 of 14 Table 3. Loss of FANCM partially suppresses the number of lethal flanking deletions that occur in the absence of BLM. F1 females with adeletion affecting the scalloped gene (left column) or causing lethality in males inheriting the deletion (right column). Parenthetical numbers indicate the total number of events analyzed: left, total number of yellow-eyed F1 females counted; right, total number of independent yellow-eyed F1 females from which F2 males were counted. The wild-type data are the same as in Table 1. a: significantly different from the wild type and fancm mutant (P < 0.00001); b: significantly different from the wild type and both single mutants (P < 0.05); Fisher's exact test.

Loss of Both HELQ and FANCM Has Additive Effects on Repair Synthesis during SDSA
The data from the gap repair assay suggest that both HELQ and FANCM play roles distinct from the BLM helicase in SDSA repair. To determine whether HELQ and FANCM may also act independently from each other during HR, we constructed helq fancm double mutants and tested them in the P{w a } assay.
While loss of either helicase resulted in a significant decrease in red-eyed progeny compared to the wild type, mutation of HELQ caused a more severe SDSA defect compared to mutation of FANCM ( Figure 5A). There was no additional significant decrease in red-eyed progeny in the helq fancm double mutant compared to the helq mutant, nor was the frequency of aborted SDSA further elevated in the helq fancm double mutant compared to the helq mutant ( Figure 5B). However, when repair synthesis in the aborted SDSA events was quantified by PCR, we observed significantly shorter repair synthesis tracts in the helq fancm double mutant compared to either single mutant ( Figure 5C and Supplemental Table S3). This suggests that HELQ and FANCM may have non-overlapping roles during SDSA. Alternatively, each protein may be limiting for a process that promotes repair synthesis.  Statistical comparisons between all mutants can be found in Supplemental Tables S1-S3.

Discussion
In this study, we characterized defects in the SDSA repair pathway in flies lacking three different DNA helicases. We confirmed reports that BLM and FANCM helicases are important for SDSA, with blm mutants affecting repair synthesis during SDSA to a greater extent than fancm mutants [23,27]. Previously, the Sekelsky lab suggested that the milder phenotypes of fancm mutants could be explained by two scenarios. First, FANCM might assist in the recruitment of the BLM-TOP3a complex to D-loop structures, where it could promote dissociation. Second, FANCM might act to unwind small D-loops that form when SDSA is initiated, but not larger D-loops that are created as SDSA proceeds [27,28]. Our examination of gap repair in blm fancm double mutants showed mostly non-additive effects of the blm and fancm mutations, consistent with the proteins having at least partially overlapping functions. However, these genetic data are not sufficient to distinguish between the two models presented above.
We also found that Drosophila HELQ helicase plays an important role in SDSA repair of mitotic double-strand breaks. HELQ was originally identified in a genetic screen for maternal effect mutants having altered eggshell morphology and was named spn-C [42]. Follow-up work showed that HELQ is needed for the repair of meiotic double-strand breaks and for resistance to the alkylating agent methyl methanesulfonate [34]. The sensitivity of helq mutants to the topoisomerase I poison topotecan is consistent with a role for HELQ in mitotic double-strand break repair. Interestingly, flies lacking either HELQ or BLM have similarly severe SDSA defects in the P{w a } gap repair assay, but the further decrease in repair synthesis observed in the double mutant suggests that they either operate independently in SDSA or are each limiting for the same function.
How might these three DNA helicases, each with 3 to 5 polarity, function to promote SDSA during gap repair? Of the three, only BLM is required to prevent deletions that occur when SDSA aborts and repair is presumably completed through an end-joining process [25]. Strikingly, the loss of either FANCM or HELQ partially suppresses the deletion-prone phenotype of the blm mutants. A possible model to explain these results could be that both FANCM and HELQ promote the early stages of SDSA. FANCM could be required to reverse unstable or unproductive D-loop structures [18] (Figure 6). In the absence of FANCM, these dead-end D-loops may need to be processed by nucleases in a way that does not create deletions flanking the original double-strand break. The synthetic lethality observed in fancm mus81 gen1 and fancm slx4 gen1 mutants lacking two structurespecific nucleases is consistent with this model [27]. When FANCM is present, productive D-loops are formed, and SDSA continues, with large and/or migrating D-loops that require BLM-TOP3α for their unwinding. In the absence of BLM, nucleases may act iteratively to process the extended D-loop, resulting in large flanking deletions [25]. This model can explain how the loss of FANCM rescues the deletion-prone phenotype of blm mutants, since SDSA intermediates that require BLM for their unwinding will not form.
a BIR-like mechanism is involved in gap repair in this system, then the helicases tested in the current study may also play a role in this type of repair.
In summary, we have shown that the BLM, FANCM, and HELQ DNA helicases are all required for efficient SDSA repair, with each likely playing a unique role in the process. The exact mechanisms by which each helicase promotes repair synthesis in the context of homologous recombination, and how these mechanisms interface with their other known functions in DNA repair and replication should be the focus of future studies. What might HELQ be doing to promote SDSA? One possibility is that it could act to enlarge the D-loop, or it may be required for the transition to a mobile D-loop as repair synthesis proceeds ( Figure 6). Indeed, the unwinding activity of mammalian HELQ is stimulated by the presence of RAD51, which is present at the advancing end of the D-loop [43]. In the absence of HELQ, perhaps only small or non-mobile D-loops are formed, resulting in limited repair synthesis. These D-loops would not require BLM for their dissociation, which is consistent with the suppression of deletions seen in helq blm double mutants.
Alternatively, HELQ might be crucial for the strand annealing stage of SDSA after the D-loop is unwound. Two recent studies identified a role for C. elegans and mammalian HELQ in annealing complementary ssDNA strands during microhomology-mediated end-joining and homologous recombination [43,44]. Drosophila HELQ has also been implicated in single-strand annealing repair [45]. While a putative function for HelQ in strand annealing during SDSA can potentially explain the decrease observed in the SDSA + LTR repair events in our P{w a } assay, it is more difficult to reconcile this model with the suppression of deletions observed in helq blm mutants. Therefore, we favor a direct role in D-loop extension for Drosophila HELQ.
Our molecular analysis of the P{w a } gap repair products suggests that synthesis normally proceeds from both ends of the gap, consistent with a mechanism where each broken end invades into the sister chromatid and repair proceeds via two-ended SDSA. However, it is also possible that repair proceeds through a break-induced replication (BIR) mechanism. Indeed, we have previously shown that the PIF1 DNA helicase, which is required for BIR in budding yeast [46,47] and promotes BIR in Drosophila [48], is needed for the efficient repair of a P{w a }-induced gap in the absence of the POL32 protein [49]. If a BIR-like mechanism is involved in gap repair in this system, then the helicases tested in the current study may also play a role in this type of repair.
In summary, we have shown that the BLM, FANCM, and HELQ DNA helicases are all required for efficient SDSA repair, with each likely playing a unique role in the process. The exact mechanisms by which each helicase promotes repair synthesis in the context of homologous recombination, and how these mechanisms interface with their other known functions in DNA repair and replication should be the focus of future studies.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/genes13030474/s1, Table S1: P-values of genotypic comparisons for SDSA + LTR annealing repair events; Table S2: P-values of genotypic comparisons for aborted SDSA repair events; Table S3: P-values of genotypic comparisons for repair synthesis tract lengths.