Collaboration between homologous recombination and non-homologous end joining in repair of meiotic double-strand breaks in Drosophila

1 Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC 27599 USA. 2 Department of Pediatrics, Division of Medical Genetics, University of Washington, Seattle, Washington and Seattle Children's Hospital, Seattle, WA. 3 Department of Biology, University of North Carolina, Chapel Hill, NC 27599 USA. 4 Integrative Program in Biological and Genome Sciences, University of North Carolina, Chapel Hill, NC 27599 USA.


Introduction
Crossovers promote the accurate segregation of homologous chromosomes during meiosis. The formation of crossovers begins with the introduction of doublestrand breaks (DSBs) by the highly conserved endonuclease Spo11 and its associated proteins (reviewed in Lam and Keeney 2014). Meiotic DSB repair differs from mitotic DSB repair in several important ways. First, DSB repair in mitotically proliferating cells can employ several repair strategies, including non-homologous end joining (NHEJ), various homology-directed pathways, and DNA polymerase theta-mediated end joining (reviewed in Ranjha et al. 2018). In contrast, DSBs made during meiosis are, under normal circumstances, repaired exclusively by homologous recombination (HR). NHEJ begins with binding of the Ku heterodimer to DNA ends, thus it has been suggested that the bound Spo11 enzyme also blocks NHEJ by preventing binding of Ku (Garcia et al. 2011, Shibata et al. 2018). An early step in HR is 5'-to-3' resection resulting in long, single-stranded 3' overhangs that also block NHEJ (Joyce et al. 2012, Lemmens et al. 2013, Yin and Smolikove 2013, Girard et al. 2018. A second key difference between mitotic and meiotic DSB repair is that the sister chromatid is typically used as a template for HR during mitotic repair but in meiosis the homologous chromosome is used. Bias for using the homolog is established by the presence of the meiotic chromosome axis and meiosis-specific recombination enzymes (Niu et al. 2005, Zanders et al. 2011, Hong et al. 2013. The axis, made up of cohesins and meiosis-specific proteins, organizes the chromosome into an array of loops which later serve as the base of the synaptonemal complex (SC) that joins homologous chromosomes together (reviewed in Loidl 2016).
Finally, mitotic HR is regulated to avoid generation of reciprocal crossovers but in meiosis reciprocal crossing over is essential and is actively promoted at selected DSB sites , the outcome of HR must, at selected sites (reviewed in Andersen and Sekelsky 2010). Complexes specific to meiotic recombination, including the ZMM proteins found in many eukaryotes (reviewed in Pyatnitskaya et al. 2019) and the mei-MCM complex of flies (Kohl et al. 2012), block non-crossover pathways and/or promote crossing-over.
When meiotic recombination is disrupted by mutations in key genes, alternative repair pathways are activated to ensure that all DSBs get repaired. If HR is blocked at early steps, NHEJ and other pathways can intervene (Joyce et al. 2012, Lemmens et al. 2013, Yin and Smolikove 2013, Girard et al. 2018, Macaisne et al. 2018. If later steps in the meiotic crossover pathway are compromised, mitotic-like HR mechanisms take over (reviewed in Kohl and Sekelsky 2013).
Another interesting question is what happens when recombination pathways are intact but a homologous chromosome is not available to use as a template. In budding yeast and Arabidopsis, meiosis in haploids or when one chromosome is monosomic reveals that HR can be used as a repair template, though less efficiently (de Massy et al. 1994, Goldfarb and Lichten 2010, Cifuentes et al. 2013.
We describe here a novel pathway for repair of meiotic DSBs when a homolog is not present. Hatkevich et al. (2019) recently demonstrated that in Drosophila melanogaster females with the separation-of-function mutation Mcm5 A7 , chromosome pairing is established normally but is lost as the during assembly of the SC, resulting in heterosynapsis (SC between non-homologous chromosomes). Previous studies had found that DSBs appear and disappear with normal kinetics in Mcm5 A7 mutants (Lake et al. 2007). Our studies show that DSB repair in these mutants is attempted first HR, but is then completed by NHEJ. Remarkably, whole-genome sequencing fails to find deletions predicted to be produced by NHEJ, indicating precise repair. We propose a model in which Spo11-bound oligonucleotides annealed to the ends of resected DSBs can function as primers for synthesis to fill in resected regions, allowing error-free repair by NHEJ. Recent studies provide evidence for a similar intermediate in mouse meiosis (Paiano et al. 2020, Yamada et al. 2020, suggesting that this may be a conserved mechanism for repair of meiotic DSBs when the homologous chromosome is unavailable.

Results and Discussion
Reduction of crossovers and loss of crossover patterning in Mcm5 A7 mutants. Mcm5 A7 is a missense, separation-of-function mutation recovered in a screen for mutants causing high levels of meiotic nondisjunction (Lake et al. 2007). The high level of nondisjunction observed in Mcm5 A7 mutants was attributed to a 90% reduction in meiotic crossovers on the X chromosome. The decrease in crossing-over was nonuniform with the interval spanning the centromere being decreased by only 80%. We extended this analysis to an autosome by scoring crossovers along 2L and observe an overall decrease of about 75%. However, the nonuniformity is more pronounced as one interval (b to pr) exhibits 150% of crossovers compared to wild-type flies ( Figure 1A, Table S1). Notably, in wild-type flies this interval accounts for about 10% of the crossovers on 2L, while in Mcm5 A7 mutants more than half of all crossovers are in this region.
Crossover distribution is a product of meiotic spatial patterning phenomena, especially crossover interference. Interference is observed as a decrease in the frequency of double crossovers from the number expected if crossovers are independent of one another (reviewed in Berchowitz and Copenhaver 2010). Because crossover distribution is perturbed in the Mcm5 A7 mutant, we asked whether interference is affected. Due to the strong reduction in crossovers in the Mcm5 A7 mutant, there is only one pair of adjacent intervals in our dataset with enough crossovers to estimate interference: dp to b and b to pr (Table S1). In wild-type flies, the incidence of double crossovers in these two intervals is less than half the number expected if they are independent (Hatkevich et al. 2017). In Mcm5 A7 mutants, however, six double crossovers were expected and seven were observed, consistent with a partial or complete loss of interference. The SC has been suggested to be important for achieving interference (Berchowitz andCopenhaver 2010, Zhang et al. 2018). The effect on interference may be explained by the presence of heterosynapsis in Mcm5 A7 mutants (Hatkevich et al. 2019); we hypothesize that discontinuities in the SC (i.e., heterosynapsis to homosynapsis) impede interference. The higher incidence of crossovers in the b to pr interval is intriguing. In Mcm5 A7 mutants, chromosome pairing appears to be normal in late leptotene is disrupted as SC assembles. The higher frequency of crossovers in the b to pr region might result from greater stability of pairing in this region.
Homologous recombination is severely reduced in Mcm5 A7 mutants. Because the number of meiotic DSBs in Mcm5 A7 mutants is normal (Lake et al. 2007, see below), the reduction in crossovers in Mcm5 A7 mutants likely reflects either a direct role for Mcm5 in the crossover pathway or a reduced ability to use the homologous chromosome for HR. In wild-type females, about a third to a quarter of DSBs are repaired as crossovers, with the remainder becoming non-crossovers; only crossovers would be reduced if these mutants have a crossover-specific defect, but both crossovers and noncrossovers would be reduced if the defect is in accessing the homologous chromosome. We used wholegenome sequencing to measure both crossovers and non-crossovers in individual offspring from Mcm5 A7 females. Since only one of four chromatids goes into the oocyte, only half of the crossover and one quarter of the non-crossovers will be detected. Only those non-crossovers that are accompanied by a gene conversion tract that spans at least one polymorphism (non-crossover gene conversions; NCO-GCs) will be detected.
We whole-genome sequenced 28 individual progeny from Mcm5 A7 females, identified crossover and NCO-GC events, and compared this data to a previously published dataset of 196 individual progeny from wild-type females . Genome-wide, we find an 86% reduction in crossovers (78 expected vs 11 observed, p<0.001) and a 67% reduction in NCO-GCs (42 expected vs 14 observed, p<0.001, twotailed T-test) ( Figure 1B). A reduction in both crossovers and NCO-GCs is consistent with a general inability to complete interhomolog HR in Mcm5 A7 mutants.
Given the results with traditional crossover mapping described above, we also asked whether crossovers and NCO-GCs cluster in the genome. On 2L, three of the four crossovers observed are located within a 2 Mb span between b and pr, the region in which genetic experiments also revealed an elevated rate of exchange ( Figure 1C). Similarly, on 2R, three of the four crossovers are grouped within a 2 Mb region, and the two COs on 3R occur within 0.4 Mb of one another (no COs were recovered on 3L and only a single CO was recovered on the X). In contrast, there is no obvious clustering of NCO-GCs, which appear to be evenly distributed throughout the genome ( Figure 1C). These findings suggest that CO and NCO formation may be differentially controlled in Mcm5 A7 mutants.

Most double-strand breaks are repaired with normal kinetics in Mcm5 A7 mutants.
Lake et al. (Lake et al. 2007) found that meiotic DSBs, as marked by -H2Av foci, are created and repaired with normal kinetics in Mcm5 A7 mutants. To extend this result, we counted -H2Av foci during DSB formation and repair. Foci first appear in Region 2A of the germarium, then decrease in number as the pro-oocyte progresses through Region 2B and into Region 3, with few or no foci remaining in Region 3, by which time breaks are thought to be repaired ( Figure 2A).
Mcm5 A7 mutants have a similar number of -H2Av foci in Region 2A as wild type ( Figure 2B, 2C). Given the canonical function of Mcm5 in DNA replication (Bell and Botchan 2013), it seemed possible that some DSBs in Mcm5 A7 mutants result from problems during pre-meiotic S phase. To test this, we used a mei-P22 mutation to eliminate meiotic DSBs. -H2Av foci are nearly eliminated in both mei-P22 single The number of -H2Av foci in Regions 2B and 3 is slightly higher in Mcm5 A7 mutants than in wild-type females (p = 0.0006 and p = 0.0112, T-test, two-tailed). The mean of 10.6 foci in Region 2A likely underestimates the total number of DSBs per meiotic cell due to asynchrony in formation and repair. Estimates range from 11 to 24 based on -H2Av foci and whole-genome sequencing (Mehrotra andMcKim 2006, Miller et al. 2016). Thus, we estimate that 90-95% of DSBs made in Mcm5 A7 mutants are repaired by Region 3. Since interhomolog HR is reduced by >80% (Figure 1), these DSBs must be repaired in other ways.

Repair of most DSBs in Mcm5 A7 mutants requires both Rad51 and DNA ligase IV.
Previous studies have shown that Mcm5 A7 mutants do not exhibit an increase in intersister crossing-over (Joyce et al. 2012, Hatkevich et al. 2019 suggesting that a strong barrier to use of the sister as an HR repair template is present in regions of heterosynapsis as in regions of homosynapsis. Together, our data suggest that DSBs made in regions of heterosynapsis are repaired by one or more non-HR pathways. One possibility is NHEJ. NHEJ is normally prevented during meiotic DSB repair but can function when there are defects in HR (Smolikov et al. 2007, Joyce et al. 2012, Lemmens et al. 2013, Yin and Smolikove 2013, Girard et al. 2018, Macaisne et al. 2018. To determine whether NHEJ is responsible for meiotic break repair in Mcm5 A7 mutants, we asked whether repair is dependent on DNA ligase IV (Lig4), an enzyme both specific to and essential for NHEJ (Lieber 2010). In lig4 single mutants, -H2Av foci in Regions 2A, 2B, and 3 are similar to those in wildtype ( Figure 3A, panel 1, p = 0.8048 versus wildtype, nested t-test, two-tailed). As in wild-type and Mcm5 A7 mutants, most -H2Av foci in lig4 mutants are dependent on Mei-P22 ( Figure 3A, panel 2). These results confirm previous observations that Lig4 is not involved in normal meiotic break repair in Drosophila (Joyce et al. 2012). In lig4 Mcm5 A7 double mutants, the number of -H2Av foci in Region 2A is similar to that seen in wild-type and single mutants, but unlike Mcm5 A7 single mutants, most foci persist through Region 2B and into Region 3 ( Figure  3A, Panel 3). Most of these foci are dependent upon Mei-P22 ( Figure 3A, Panel 4). Thus, the timely repair of meiotic DSBs in Mcm5 A7 mutants requires Lig4.
The simplest interpretation of our -H2Av focus data is that there can be both homosynapsis and heterosynapsis within a single nucleus, and that DSBs in regions of homosynapsis are repaired by HR, whereas DSBs in regions of heterosynapsis are repaired by NHEJ. An important question is whether HR is initially attempted at all DSBs, with NHEJ as a backup. To address this question, we determined what proportion of DSBs in Mcm5 A7 mutants are dependent on Rad51 (in Drosophila, this is encoded by the spn-A gene; for simplicity, we refer to the Spn-A protein as Rad51). Meiotic DSBs are not repaired in spn-A mutants, resulting in a modest accumulation of foci as development proceeds ( Figure 3B). Surprisingly, Mcm5 A7 spn-A double mutants have the same number of -H2Av foci in all regions as spn-A single mutants ( Figure 3B). This implies that all breaks, regardless of whether located in regions of heterosynapsis or homosynapsis, are initially processed by HR.

NHEJ-dependent DSB repair in Mcm5 A7 mutants is precise.
Meiotic DSBs are formed from when Spo11 (mei-W68 in Drosophila; for simplicity, we refer to the Mei-W68 protein as Spo11) cleaves with 2-bp 5' overhanging ends to which the enzyme remains covalently bound (Keeney 2008) (Figure S1, a). The requirement for Rad51 to repair all DSBs most likely means that resection occurs to generate singlestranded DNA. Resection is initiated by nicks 3' of the cleavage site, then extended exonucleolytically both 5'-to-3' and 3'-to-5' (Garcia et al. 2011) (Figure S1, b). In most depictions of meiotic recombination models, the Spo11 bound-oligonucleotide (Neale et al. 2005) is released after resection ( Figure S1C), and Rad51 is loaded onto resected strands up to the 3' end ( Figure S1, d) to facilitate homology search and strand exchange ( Figure S1, e), allowing extension of the 3' end by repair synthesis to then give rise to repair of DSBs as either crossovers or NCO-GCs ( Figure S1F). However, in Mcm5 A7 mutants, all meiotic DSBs require Rad51 for repair, and about 80% of these also require Lig4. As Spn-A and Lig4 normally function in separate DSB repair pathways, the interplay between these two proteins during Mcm5 A7 mutant repair is unknown. We propose two Rad51-, Lig4-dependent mechanisms, both of which are modifications to the canonical HR repair pathway described above ( Figure S1, a-f). In the first model, when the homology search fails for DSBs in regions of heterosynapsis ( Figure S1, g), the long overhangs would need to be clipped to allow repair by NHEJ (Daley and Wilson 2005) (Figure S1, h); this would result in a large deletion ( Figure S1I). Given an average gene conversion tract length of 350-450 bp (Hilliker et al. 1994, Blanton et al. 2005, the average deletion size would be expected to be within this range if conversion tracts span the entire resected area on both sides of the DSB, or twice this size, if conversion tracts correspond to one side of a symmetrically-resected DSB. An alternative model is suggested by recent analyses of single-stranded DNA (ssDNA) during meiotic recombination. Two efforts to map ssDNA during meiotic recombination in mice found that the extreme 3' ends do not appear to be singlestranded when Rad51 loads, leading to the hypothesis that Spo11-bound DNA remains annealed until after strand exchange (Paiano et al. 2020, Yamada et al. 2020) ( Figure  S1J-M). Yamada et al. speculate that the Spo11-bound oligonucleotides can prime fill-in synthesis if the homology search fails ( Figure S1, n). If the 2-nt overhangs generated at initial cleavage were clipped by an exonuclease (Figure S1, o), repair by NHEJ would result in deletions of about 2-6 nucleotides ( Figure S1, p). Alternatively, if Spo11 was removed ( Figure S1, q), NHEJ could result in precise repair, restoring the original sequence ( Figure S1, r).
To distinguish between these models, we used the methods described in Miller (2020) to search for de novo deletions in the Mcm5 A7 whole-genome sequence dataset described above. In simulated genotypes with small deletions (1-10 bp), 85% were detected. In simulations with large deletions (1-1000 bp), 57% were detected, with no apparent bias in size within this range ( Figure S2). These methods successfully identified 20 de novo deletions of various sizes in sequences from progeny of mutants with defects in synaptonemal complex (Miller 2020). The 28 progeny we sequenced would be expected to harbor approximately 84 sites repaired by NHEJ (see Methods for calculation details). We would expect to detect 48 of these if repair produced large deletions ( Figure S1, i) or 71 if it produced small deletions ( Figure S1, p). We detected neither small nor large deletions in our dataset. We also looked for translocations or inversions that might result from joining of misappropriate ends, using methods that have been used successfully to map rearrangements in balancer chromosomes ); we did not detect any such rearrangements. Taken together, we can conclude that NHEJ is able to repair ends precisely in Mcm5 A7 mutants ( Figure  S1, r).

Conclusions.
Previous studies have found that loss of proteins required to catalyze the resection needed for HR can lead to repair of meiotic DSBs by NHEJ (Joyce et al. 2012, Lemmens et al. 2013, Yin and Smolikove 2013, Girard et al. 2018). Here, we have shown that most DSBs made in Drosophila Mcm5 A7 mutants require both Rad51 and Lig4, indicating that NHEJ can be used to repair DSBs even after resection, presumably at sites where the homologous chromosome is not available as a template. Although NHEJ is generally characterized as error-prone, in this situation it is precise, with no apparent mutations.  Figure S1 for additional models. In the canonical model (left), DSB formation involves concerted nicking by two Spo11 proteins (yellow circles), which remain covalently bound to 2-nt 5' overhangs through a tyrosyl phosphodiesterase bond (a). Resection is initiated by nicks (green arrowheads) 3' of the DSB, then (b) extended bidirectionally (Garcia et al. 2011). Rad51 and associated proteins (and in many species, Dmc1) assembles on the single-stranded DNA exposed by resection (c). Recent observations suggest that a short Spo11-oligonucleotide remains annealed at the 5' end of each strand (Paiano et al. 2020, Yamada et al. 2020)}. A homology search and strand exchange produces a D-loop (d). Removal of the Spo11-oligonucleotides, either by cleavage or dissociation, allows repair synthesis to proceed (e). Subsequent steps (not shown) produce crossover and non-crossovers. We hypothesize that if a homologous template cannot be found, such as in regions of heterosynapsis in Mcm5 A7 mutants, the Spo11-oligonucleotides function as primers for fill-in synthesis (f). NHEJ can then complete repair through annealing of the 2-nt overhangs (g), resulting in precise repair. We propose a model for precise NHEJ post-resection, based on the recent discovery that Spo11-oligonucleotides remain annealed to DSB ends after resection in mouse meiosis (Paiano et al. 2020, Yamada et al. 2020 (Figure 4). In our model, the Spo11-oligonucleotide remains annealed during the homology search (Figure 4a-c). If a homologous template is found, strand exchange produces a D-loop (Figure 4d), then the Spo11-oligonucleotide are released by dissociation of the oligonucleotide, cleavage invading strand to remove the double-stranded region, or enzymatic reversal of the tyrosyl phosphodiesterase bond, allowing repair synthesis to proceed (Figure 4e). We hypothesize that if homology cannot be found, such as in regions of heterosynapsis in Mcm5 A7 mutants, the Spo11-oligonucleotides are used as primers for fill-in synthesis (Figure 4f). The Spo11 is then enzymatically removed and NHEJ uses the 2-nt overhangs (Figure 4h) to restore the original sequence (Figure 4h).
An important unknown step in our model is how Spo11 is removed. Likely candidates include tyrosyl-phosphodiesterases (TDPs), which directly reverse protein tyrosyl-DNA complexes generated by topoisomerases (reviewed in Pommier et al. 2014), also proteins with an SprT metalloprotease domain that can remove DNA-protein crosslinks (Lopez-Mosqueda et al. 2016, Stingele et al. 2016, Vaz et al. 2016). The only TDP in Drosophila is Tdp1, encoded by the essential gene glaikit (glk) (Dunlop et al. 2000, Guo et al. 2014. Drosophila has orthologs of the SprT domain proteins SPRTN, encoded by maternal haploid (mh) (Delabaere et al. 2014), and Germ Cell Nuclear Acidic Peptidase (GCNA) (Bhargava et al. 2020). We constructed mh Mcm5 A7 double mutants but did not detect persistence of -H2Av foci ( Figure S3). Because glk is an essential gene, we generated flies with germline expression of a transgene for RNAi knockdown of glk in with Mcm5 A7 mutants; we also did not detect persistence of -H2Av foci in these females ( Figure S3). GCNA is difficult to test because mutants have Spo11independent -H2Av foci from pre-meiotic S phase, as well as germline developmental defects (Bhargava et al. 2020).
Although the identity of the protease that removes Spo11 in our model remains unknown at present, it is interesting to speculate on what the consequences of failure to remove Spo11 might be. If this enzyme is used in normal meiosis, backups might include dissociation or cleavage, as described above. If a homologous template is not detected, as in Mcm5 A7 mutants, persistence of unrepaired DSBs might result in sterility. Alternatively, a backup might be endonucleolytic cleavage of the 2-nt overhangs ( Figure  S1, p) would allow repair by NHEJ, though it would be imprecise.
In summary, our experiments reveal the existence of a novel backup mechanism to repair meiotic DSBs when an HR template cannot be found. This repair mechanism involves repair by NHEJ that, even though resection apparently occurs, is precise. We propose that this is possible because of retention of Spo11-bound oligonucleotides at the ends of the DSBs. Our results, coupled with the discovery that Spo11-oligos remain anneal in mouse meiosis (Paiano et al. 2020, Yamada et al. 2020, suggests that this backup pathway may be conserved, at least in animals.

Materials and Methods
In all immunofluorescent and genetic experiments, Drosophila melanogaster adult females 3-10 days old were used. In whole genome sequencing studies, individual male progeny were used. Flies stocks were maintained on standard medium at 25°C. In this article, Drosophila nomenclature was generalized for readership. For transparency, genotypes, specific alleles, and associated figures are listed in the Genetic assays. X chromosome NDJ was evaluated by scoring progeny from virgin females of the desired genotype crossed with y cv v f / T(1:Y)B S males. Viable exceptional XXY females have Bar-shaped eyes, and viable exceptional XO males have wild-type eye shape and express the y cv v f mutant phenotypes. Crossovers on chromosome 2L were measured by crossing virgin net dpp d-ho dpy b pr cn / + females of the desired genotype to net dpp ho dp b pr cn males. Vials of flies were flipped after three days of mating. Resulting progeny were scored for all phenotypic markers.
Dissection and immunofluorescence (IF) of whole mount germaria. Ten three-to five-day old virgin females were fattened overnight with yeast paste in vials with ~5 males of any genotype. Ovaries were dissected in fresh 1x PBS and incubated in fixative buffer for 20 minutes. Fixative buffer: 165 µL of fresh 1x PBS, 10 µL of N-P40, 600 µL of heptane, and 25 µL of 16% formaldehyde. After being washed 3 times in 1x PBS + 0.1% Tween-20 (PBST), ovaries were incubated for 1 hour in 1 mL PBST + 1% BSA (10 mL of PBST + 0.1 g BSA). Ovaries were incubated overnight in 500 µL primary antibody diluted in 1 mL PBST + 1% BSA at 4 C on a rocking nutator. Ovaries were then washed 3x in PBST and incubated in 500 µL secondary antibody diluted at 1:500 in PBST + 1% BSA for 2 hours under foil. Ovaries were mounted in 35 µL of ProLong Gold + DAPI on a microscope slide using fine-tip forceps to spread ovaries.
Antibodies for C(3)G (Anderson et al. 2005) and -H2Av (Rockland) were used. Images of whole mount germaria were taken Zeiss LSM880 confocal laser scanning microscope using 63x/0.65 NA oil immersion objective, with a 2x zoom using ZEN software. Images were saved as .czi files and processed using FIJI (Schindelin et al. 2012). DSBs were quantified as described below.
For quantification of -H2Av foci, FIJI (Schindelin et al. 2012) was used to visualize images, and contrast and brightness were adjusted for optimal viewing. In each region of the germarium, individual -H2Av foci were manually counted in nuclei expressing C(3)G. Data are represented as mean  95% CI, and the two tailed T-test or nested Ttest was used to determine significance. Genotypes were blinded to the scorer.
Whole genome sequencing, alignment, and SNP calling. Mcm5 A7 was crossed into a y cv v f line with isogenized chromosomes X and 2 (iso1). Df(3R)Exel7305 was crossed into a w 1118 line with isogenized chromosomes X and 2. These two lines were crossed to one another and resulting females (heterozygous for iso1 and w 1118 on X and 2 and Mcm5 A7 /Df(3R)Exel7305 on 3) were backcrossed with iso1 males. Resulting male progeny were used for whole-genome sequencing.
DNA was isolated from individual male progeny using the Qiagen Blood and Tissue Kit and sheared using a Covaris S220 sonicator. KAPA HTP Library Prep kit (KAPA Biosystems, Cat. No. KK8234) was used to construct libraries with NEXTflex DNA barcodes (BiooScientific, Cat No. NOVA-514104). Libraries were pooled and run on an Illumina NextSeq 500 as 150 bp paired-end samples using the high-output mode. Real Time Analysis software version 2.4.11 and bcl2fastq2 v2.18 demultiplexed reads and generated FASTQ files. FASTQ files were aligned to version 6 of the Drosophila melanogaster reference genome (dm6) using bwa mem version 0.7.17-r1188 with default settings (Li and Durbin 2010). SNPs were called using both Samtools (Li et al. 2009) andGATK HaplotypeCaller (McKenna et al. 2010). Depth of coverage analysis revealed two X0 males (mcm05-01 and mcm05-12).
Detection of CO and NCO-GC events and calculation of expected events. CO and NCO-GC events were identified as in Miller et al. (2012). Briefly, for each individual offspring sequenced, the VCF file was used to identify changes in polymorphisms from one parent to another. SNP density per chromosome arm was: 1/531 bp for the X, 1/255 bp for 2L, 1/254 bp for 2R, 1/249 bp for 3L, and 1/318 bp for 3R. Each candidate change was validated visually using IGV. A total of 28 individual males were sequenced to revealed 11 COs and 16 NCO-GCs. 11 NCO-GCs were validated using PCR and Sanger sequencing.
Expected CO and NCO-GC events were calculated based on wild-type values from  in which 541 CO and 294 NCO-GC events were observed in 196 wild-type meioses. Fisher's exact test (two-tailed) was used to determine significance between observed and expected event frequencies (GraphPad QuickCalcs; http://www.graphpad.com/quickcalcs/).

Identification of deletions and generation of simulated deletions.
Multiple approaches were used to search for deletions. First, VCF files generated by Samtools and GATK HaplotypeCaller were searched for unique deletion polymorphisms. Candidate deletions were visually examined using IGV (Robinson et al. 2017). Second, Pindel v0.2.5b9 (Ye et al. 2009) and Breakdancer v1.1_20100719 (Fan et al. 2014) were used to identify larger deletions and a custom script was used to parse the output and identify novel events. No novel deletions were identified using both approaches.
To validate our approach, simulations were done as in Miller (2020). Briefly, 200 simulated genomes were created based on the dm6 reference genome, 100 with deletions of 1-20 bp, and 100 with deletions varying from 1-1,000 bp. SNPs were randomly placed approximately 1 every 500 bp in these synthetic genomes. For each genome a minimum of five and maximum of 25 deletions were made. Each deletion was randomly assigned a size, a position on X, 2, or 3, and a one of the four haplotypes corresponding to the four meiotic products; one of these haplotypes was selected as the offspring. A second synthetic genome representing the male haplotype was also generated with no X but with a Y chromosome, with SNPs randomly placed approximately 1 every 500 bp. Using each synthetic genome as a reference, ART (Huang et al. 2012) was used to generate 100 bp paired-end reads at approximately 10x coverage with an average insert size of 400 bp for both the male and female synthetic genomes. FASTQ files were then combined into a single FASTQ and aligned to dm6 using bwa. These synthetic genomes were analyzed similar to the real ones, with SNPs called using both Samtools and GATK HaplotypeCaller, and Pindel and Breakdancer used to identify larger deletions. Analysis of vcf files from simulated genomes revealed that the approaches described above identify >95% of novel deletions of 1-20 bp and at least 65% of novel deletions 1-1,000 bp.
To calculate the number of deletions we expected to recover we used a conservative assumption of 15 DSBs per meiosis. Based on the whole-genome sequencing data, HR is decreased by ~80% in Mcm5 A7 mutants, suggesting that NHEJ repairs at least 12 DSBs per meiosis; each oocyte receives one of four chromatids, resulting in an average of three deletions per progeny. Therefore, we expect 84 deletions in our sample size of 28 meioses. Based on the simulated genomes described above, we should detect 71 of these if they are small, 48 if they are large.
Genetic distances are expressed in centiMorgans (cM), calculated by 100 * (R / n), where R is the number of recombinant progeny in a given interval (including single, double, and triple crossovers), and n is the total number of progeny scored. 95% confident intervals were calculated from variance, as in Stevens [43]. Molecular distances (in Mb) are from the positions of genetic markers on the Drosophila melanogaster reference genome, release 6.12 (Thurmond et al. 2018). Crossover density (or frequency), as calculated by cM/Mb, exclude transposable elements (Hatkevich et al. 2017).
Illumina data used in this project has been deposited at the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) under project PRJNA623030. Code used in this project are available at: https://github.com/danrdanny/mcm5-A7.