Dm brca2 in DNA Repair and Checkpoints Figure 1. brca2 Mutations Dramatically Decrease HR-h Repair, Increase SSA Repair, and Result in Increased Deletion Formation (A) The Rr3 assay measures the relative repair pathway usage for repair of I-SceI endonuclease-cut breaks in the pre-meiotic germline of Drosophila males. The Rr3 transgene contains an I-SceI site flanked by two partial copies of dsRed. One dsRed copy contains a 147 bp duplication. Repair by SSA reconstitutes dsRed and results in red fluorescent progeny. Repair by NHEJ modifies the I-SceI site and results in progeny that are dark regardless of the presence of the endonuclease. The EJ1 transgene is a variant of Rr3 which has a non-functional I-SceI site and a 16 bp deletion 156 bp from the I-SceI site. Progeny resulting from repair by HR-h are also dark even when the endonuclease is present and can be distinguished from NHEJ by PCR. PCR analysis also distinguishes between short and long tract HR-h; long-tract HR-h results in the inclusion of the deletion on EJ1 and a smaller product. Deletions are measured by the loss of the wþ marker. (B) In Cross 2, the EJ1 chromosome was present, and repair by HR-h could occur. Error bars represent the standard errors between individual males; P- values were calculated using a permutation test described in [31]. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 72/3861, 72/3163, and 78/3486. (C) In Cross 1, the EJ1 chromosome was not present, and repair by HR-h was not available. Number of males/total Rr3-carrying progeny counted for OreR, brca2, and okra, respectively: 66/7103, 67/6054, and 65/5546. aThe deletion class was not isolated prior to sorting and therefore represents a small portion of the NHEJ class. doi:10.1371/journal.pgen.0040031.g001 (Figure 1C). In cross 1, the use of SSA increased to 111% of wild-type levels and NHEJ decreased to 74% percent of wild- type levels in brca2 mutants. It is possible to interpret this result as a partial requirement for brca2 in NHEJ. However in light of similar results with other HR-h defective mutants [32], our similar results for okra mutants (Figure 1C), and the fact that we did not see a decrease in NHEJ in cross 2 (P ¼0.50, Figure 1B), we favor the explanation that changes in NHEJ and SSA levels in cross 1 were due to defects in HR-s that were indirectly measured by changes in NHEJ and SSA. Even though HR-s was not measured, HR-s repair is likely initiated in brca2 mutants. If DSBs that abort HR-s in brca2 and okra mutants in cross 1 are shuttled speciﬁcally into the SSA pathway like the aborted HR-h DSBs in brca2 and okra mutants in cross 2, then the effect would be to increase SSA relative to NHEJ in cross 1. Similar arguments have been proposed for the effects seen in spnA mutants [32]. In conclusion we found that brca2 is required for HR-h and that the loss of HR-h is associated with an increase in the use of SSA repair. In addition, the rate of deletion formation near DSBs increases in brca2 mutants. These changes in DSB repair represent a shift towards more mutagenic repair of DSBs in brca2 mutants that are similar to changes seen in spnA/rad51 and okra/rad54 mutants ([32] and Figure 1). There- fore we do not ﬁnd evidence for a Rad51-independent role for Drosophila Brca2 in somatic DNA repair. Failure to Repair Meiotic DSBs in brca2 Mutants Leads to Partial Activation of the Meiotic Recombination Checkpoint Mutations in genes required for repair of meiotic DSBs, such as rad51/spnA, cause female sterility due to activation of a meiotic checkpoint that affects dorsal-ventral patterning of the eggshell and embryo [1,14]. The DNA endonuclease Mei- W68 catalyzes DSB formation in the germarium of the Drosophila ovary as the initiating step in meiotic recombi- nation [33]. If the DSBs persist, such as in DNA repair mutants, a checkpoint is activated that results in the inefﬁcient translation of Gurken, the ligand for Epidermal PLoS Genetics | www.plosgenetics.org 0004 February 2008 | Volume 4 | Issue 2 | e31