Figure 5. Brca2 Co-Immunoprecipitates with Rad9, a Member of the 9-1- 1 Checkpoint Complex Western blots from FLAG-Rad9 immunoprecipitations probed for FLAG and HA epitopes. Approximately 1/20 of the lysate prior to immuno- precipitation and the entire immunoprecipitated protein fraction was loaded. Ovaries from females carrying single copies of PfUAS-Brca2-HAg and/or PfUAS-FLAG- Rad9g and the germline driver nanos-Gal4-VP16 were harvested for the immunoprecipitation. doi:10.1371/journal.pgen.0040031.g005 observed a slight increase in the number of mitotic nuclei in hus1 mutants relative to wild-type after low dose irradiation, we still observe a 4-fold decrease in the number of mitotic cells after IR as compared to unirradiated discs, indicating that the G2/M checkpoint is largely intact in hus1 mutants after low dose IR. Brca2 Interacts with Rad9 As brca2 and hus1 mutants are both required for efﬁcient activation of the meiotic recombination checkpoint but are not required for the irradiation checkpoints, we wondered if Brca2 acted in concert with the 9-1-1 complex to enforce checkpoint control. We found that epitope-tagged Rad9, a member of the 9-1-1 complex, and Brca2 expressed in the germline cells of the Drosophila ovary co-immunnoprecipi- tated (Figure 5). Immunoprecipitations for FLAG-tagged Rad9 pulled down HA-tagged Brca2. We roughly estimate from 3 independent experiments that between 1/40 (data not shown) to ; 1/400 (Figure 5) of the total HA-Brca2 protein was co-immunnoprecipitated by FLAG-Rad9. It could be that only a subset of the Rad9 present in all regions of the Drosophila ovary binds Brca2. Alternatively, the interaction between Brca2 and Rad9 may be stage-speciﬁc, only occur- ring in the region of the germarium in which recombination intermediates are present and in which a transient check- point response may occur. The physical interaction of Brca2 and Rad9 and the similarities in genetic requirements for hus1 and brca2 in Drosophila and mammalian checkpoints strongly suggest a common role for Brca2 and the 9-1-1 complex in checkpoint function. Discussion Drosophila Brca2 as a Model for Human BRCA2 Our results demonstrate that the predicted gene CG31069 is a functional BRCA2 homolog required for meiotic and mitotic homologous recombination. We have made mutations in Drosophila brca2 in two distinct genetic back- grounds and rescued the ovarian defects by genomic rescue. Dm brca2 in DNA Repair and Checkpoints Unlike in mammals in which mutations are early embryonic lethal [50], Drosophila brca2 mutants are homozygous viable, possibly because of the long period of maternal gene expression during Drosophila embryonic development. It is still uncertain whether Drosophila Brca2 contains a cryptic DNA binding domain similar to mamma- lian BRCA2 or if this function is encoded in a tightly regulated interacting protein. Further biochemical studies will be necessary to resolve this question, but it is clear from our functional analysis of the role of Brca2 in DNA repair, as well as a recently reported physical interaction with SpnA/ Rad51[51], that Drosophila brca2 represents a functional homolog of the human breast cancer susceptibility gene. Clearly, due to the viability of Drosophila brca2 mutants and the power of Drosophila genetics, Drosophila offer a promising new opportunity for uncovering novel roles for BRCA2 during development. In this work we presented a thorough characterization of the role of Brca2 in DSB repair and we uncovered a novel function for Brca2 in the meiotic recombination checkpoint. Drosophila Brca2 and Repair Using the Rr3 assay [30] we showed that in brca2 mutants, DSB repair is shifted towards repair by potentially mutagenic repair pathways. Repair by homologous recombination is dramatically decreased in brca2 mutants, and repair by single- strand annealing predominates. Repair by SSA always results in the loss of the sequences between annealed repeats. SSA repair is restricted to DSBs ﬂanked by repetitive elements, though due to the highly repetitive nature of higher eukaryotic chromosomes, SSA repair can represent a signiﬁcant source of mutagenesis in higher eukaryotes. Our results contrast with studies in C. elegans, in which indirect in vivo experiments and in vitro annealing experiments[16,25] have lead to the suggestion that the C. elegans BRCA2 homolog is required for SSA repair. Our results are more similar to the effects seen in mammalian cell culture experiments with hypomorphic BRCA2 mutations, in which decreases in HR repair correlated with increases in SSA repair [26–29]. In addition, Brough et al. (2008) also have recently reported a similar inverse relation- ship between SSA and HR-h in a Drosophila brca2 mutant using a simpliﬁed DSB repair assay [51]. In the Rr3 assay when one pathway is compromised, the sum of the relative pathways usage in these mutants still equals near 100%, even though the percentages are calcu- lated from different populations and are not forced to equal 100% ([32] and our own work). This observation plus the fact that different effects on repair pathway balance have been observed among mutants with decreases in the same pathway suggest that regulated compensation can occur. For example, mutations in mus101 and mei-41 both result in a decrease in SSA, but the former are compensated by increases in NHEJ and HR-h, while the latter is compensated by NHEJ only [32]. In cross 2, we saw a signiﬁcant increase in the use of the SSA (P ,1e 8), but no signiﬁcant difference in the relative level of NHEJ (P ¼ 0.50) in brca2 mutants (Figure 1B), indicating compensation by the SSA pathway in brca2 mutants. Compensation of decreases in HR-h by the SSA pathway seems to be a common response to deﬁciencies in genes required for HR-h. In mammalian cell culture studies, Stark et al. (2004) found a similar inverse relationship between SSA and HR in Brca2 and Rad51 mutants, while Brca1 mutants had PLoS Genetics | www.plosgenetics.org 0008 February 2008 | Volume 4 | Issue 2 | e31