decreases in both SSA and HR [29]. Using the Rr3 assay, Johnson-Schlitz et al. (2007) found that the signiﬁcant decreases in HR-h in Drosophila dmBlm, top3a, and spnA mutants were compensated entirely through increases in SSA, while in okra mutants compensation occurred through signiﬁcant increases in both SSA and NHEJ. In our experi- ments we observed compensation in okra mutants entirely through SSA, and the exact cause of the discrepancy is unclear. Possibly the discrepancy lies in the use of different endonuclease sources or in different combinations of okra alleles used (okraAA/RU versus okraRU/WS), though mutagen sensitivity studies have suggested that the AA and WS alleles are of similar strength [40]. Regardless, it is clear that brca2 mutants are compensated by SSA, similar to most mutants deﬁcient for HR-h in Drosophila and other organisms. It is also notable that short-tract HR-h is more strongly affected in brca2 mutants than long-tract HR-h. Short-tract HR-h was decreased 64-fold relative to wild-type values and long-tract HR-h was decreased 9-fold (Figure 1B). The residual HR-h repair probably reﬂects repair that occurred early when low levels of maternal Brca2 were still present and wild-type levels of Brca2 may be required to restrict the extent of gene conversion during HR-h repair. Gene conversion tract length during HR-h repair has important implications in maintaining genomic integrity as the poten- tial for loss of heterozygosity increases with increasing tract length. Mutations in okra led to a similar shift towards long- tract HR-h (Figure 1B), and homozygous and heterozygous mutations in spnA have also been observed to alter the balance of HR-h in towards long-tract HR-h [32]. These results suggest that reductions in levels of the enzymes required for strand invasion can result in increased rates of loss of heterozygosity during HR-h repair. Increased long- tract HR-h repair relative to short-tract HR-h repair in brca2 mutants may represent increased rate of DNA synthesis, increased resection prior to strand invasion, increased stability of recombination intermediates, unequal repair of the heteroduplex DNA, or a combination of these processes. In mammalian cells, an increase in the extent of gene conversion has been observed in both Rad51K133R and Xrcc3, a Rad51 paralog, mutants; though these observed increases are thought to have arisen from different mechanisms due to differences seen in gene conversion tract continuity [29,52]. Models explaining the increase in the inclusion of the 16 bp deletion in Drosophila brca2 and spnA mutants would ﬁrst need to determine whether conversion tracts in these mutants are continuous or discontinuous by using a more complicated reporter design. Drosophila Brca2, the 9-1-1 Complex, and Checkpoints We found a novel requirement for brca2 in transduction of the meiotic recombination checkpoint signal. Initially the meiotic phenotypes of brca2 mutants were surprisingly different from the spindle class mutants previously studied. First, even with the exacerbation of the ventralization defect by growth at 25 8C, the eggshell ventralization phenotype of brca2 mutants was signiﬁcantly weaker in spite of similar levels of persistent DSBs (Figure 2). Second, the kinetics of the eggshell phenotype were opposite to the kinetics of classical spindle mutants. In spnA,B,C,D and okra mutants the pheno- type is weak during the initial days in which the females are fed yeast, but after 5–7 days on yeast spindle mutants lay Dm brca2 in DNA Repair and Checkpoints predominately severely ventralized eggs. In brca2 mutants, ventralized eggs were only reliably laid during the ﬁrst 1–4 days on yeast (data not shown). Given our results, it is now clear that the requirement for brca2 in efﬁcient transduction of the checkpoint signal masks the strong eggshell ventraliza- tion phenotype that is normally suggestive of a role in meiotic DSB repair. As an increasing number of proteins with dual roles in DNA repair and checkpoint function are being identiﬁed, it will be interesting to see if there are additional dual function proteins functioning in Drosophila meiosis. While the classical meiotic repair mutants have strong oogenesis phenotypes, weak or absent eggshell patterning defects may not preclude a role in meiotic DNA repair if coupled to a role in checkpoint transduction. We found that the ovarian phenotypes of brca2 mutants were suppressed by mutations in chk2, but were not sup- pressed by mei-41 mutations. This ﬁnding is in contrast to the other spindle class mutants which are suppressed by both mei- 41 and chk2 mutations [1,14,39]. Our model is that in females with an intact checkpoint response, the checkpoint activation is dependent upon mei-41 and chk2. However, because Brca2 acts in a similar step in the checkpoint pathway as Mei-41, no additional suppression is observed in the mei-41; brca2 double mutant. Since both the eggshell and karyosome defects of brca2 mutants can be suppressed by chk2 it is probable that the residual checkpoint activation in brca2 mutants is due to activation of Chk2 by the upstream checkpoint kinase Atm. It is not currently possible to test the involvement of Atm in the checkpoint as even viable, hypomorphic atm single mutants have eggshell and karyosome defects. There is however evidence that Mei41-independent checkpoints exist in Dro- sophila meiosis. Upregulation of transposable elements in the Drosophila germline, as seen in cutoff mutants, results in eggshell patterning defects that can be suppressed by chk2 but not by mei-41 mutations [53]. The checkpoint activated in cutoff mutants is, however, distinct in at least some aspects from the checkpoint activated in brca2 mutants. Unlike in brca2 mutants, the checkpoint activated in cutoff mutants results in a loss of germline cells as well as eggshell patterning defects, and these defects cannot be suppressed by mutations that prevent DSB formation. According to our model, Mei41-independent checkpoint activation in brca2 mutants is strong enough, with respect to either signal strength or signal duration, to result in a karyosome defect but not strong enough to result in strong eggshell patterning defects. Clearly Mei-41 is responsible for the bulk of checkpoint activation in classical spindle mutants but it remains possible that Atm may play a supporting role. It is notable that the classical spindle phenotypes are typically scored after 5–7 days on yeast at ambient temperature and that at these conditions the brca2 single mutant phenotype was very weak (data not shown). Therefore it is possible that under the conditions used in this study mei-41 mutations may also not completely suppress the classical spindle mutations. In interpreting the role of brca2 in the transduction of the meiotic recombination signal we have had to focus on the eggshell phenotype as brca2 single mutants have karyosome defects. This phenotype is similar to that seen in hus1 mutant females. hus1 mutants are able to suppress the eggshell defects of spindle mutants but do not suppress the karyosome defects of the spindle mutants as hus1 single mutants have karyosome defects [36]. Though it has not been tested in this study, it PLoS Genetics | www.plosgenetics.org 0009 February 2008 | Volume 4 | Issue 2 | e31