seems possible that, similar to brca2 mutants, the hus1 mutant karyosome defect is a result of persistent DSBs and partial activation of the checkpoint. We also found that Brca2 co-immunoprecipitated with Rad9. The 9-1-1 complex forms a heterotrimeric ring that is loaded onto resected single-stranded DNA ﬂanking a DSB following replication stress [54,55]. The 9-1-1 complex associates with DSBs independent of ATR, and mediates Chk1 phosphorylation by ATR through interaction with TopBP1[56–59]. The 9-1-1 complex may or may not use a similar mechanism to activate Chk2 during the meiotic recombination checkpoint. Although the precise functional relevance of the Rad9-Brca2 interaction remains to be explored, our co-immunoprecipitation results further suggest that the role of Brca2 in checkpoint control is upstream of Chk1/Chk2. Research in mammals and our work in Drosophila has shown that the 9-1-1 complex and BRCA2 are speciﬁcally required for the checkpoints thought to be activated in response to large stretches of single-stranded DNA. In Drosophila we have shown that they are required for the meiotic recombination checkpoint, but not the irradiation checkpoints. In mammals, BRCA2 and the 9-1-1 complex are required for several S-phase checkpoints [3,10,49]. Now that a physical connection between Rad9 and Brca2 has been observed, it will be interesting to determine the degree of functional overlap between Brca2 and the 9-1-1 complex in the DNA damage responses. In light of the absence of a predicted DNA binding domain in Drosophila brca2 it is tempting to predict that the Drosophila 9-1-1 complex and Brca2 may have a common role in both repair and checkpoints. While the functional overlap is complete for the checkpoints examined to date, it is clear from mutagen sensitivity assays that the functional overlap between 9-1-1 and Brca2 in DNA repair is not absolute. hus1 mutants are severely sensitive to MMS, but not to IR, while brca2 mutants are moderately sensitive to MMS and severely sensitive to IR ([36] and this study). In conclusion we have found that the Drosophila homolog of BRCA2 is required for mitotic and meiotic homologous recombination and in the absence of brca2 error-prone repair predominates. brca2, similar to the 9-1-1 complex with which it physically interacts, has a second requirement during meiosis in the activation of the meiotic recombination checkpoint but is not required for checkpoints that respond to irradiation induced damage, indicating a specialized role for brca2 in checkpoint control. Methods Fly strains and transgenes. Flies were maintained at 25 8C. Additional information about culturing conditions can be found in Text S1. brca256E was isolated by screening for imprecise excisions after mobilization of P SUPor-PgCG30169KG03961 which was inserted f in the 5’UTR of CG30169. brca256E removes the majority of the coding sequence of CG30169 and most of the 5’UTR of CG4612; the sequence junction is GTGGTGGCGGCT/ AACTTGCCGGCAA. brca2KO was created by ends-out homologous recombination using the targeting vector pW25 [21]. Flanking homologous sequences were ampliﬁed, inserted into the BsiWI/AscI and Acc65I/SphI sites of pW25, and predicted open reading frames were veriﬁed by sequencing. The sequence junction of the deleted region is: TTGTTTTGGAAATGC/ AAGGTGCCCACTTAC. The structure of the resulting knock-out was veriﬁed by PCR and Southern blot analysis (data not shown). To make PfCG30169g a 5.8 Kb XhoI-NotI fragment containing CG30169 was Dm brca2 in DNA Repair and Checkpoints isolated from the BAC clone BACR11c07 (BACPAC Resource Center) and ligated into pCasper4. To make PfUAS-Brca2-HAg the region containing the coding sequence of Brca2-PA was PCR-ampliﬁed using primers that created a 5’ NotI site and a 3’ XbaI site. Three copies of HA followed by BglII and KpnI sites were placed downstream of the XbaI site. The resulting NotI-KpnI fragment was cloned into pUASp. To make the PfUAS-FLAG- Rad9g, the coding sequence of Rad9-PA was ampliﬁed using primers to create a 5’ KpnI site and a 3’ XbaI site. The FLAG peptide sequence was added at the 5’ end following the KpnI site. The resulting KpnI -XbaI fragment was cloned into pUASp. nanos-Gal4-VP16 driven Brca2-HA expression can rescue the eggshell patterning defects of brca2 mutants (data not shown). P-element- mediated germ-line transformation was carried out according to standard protocols [60]. The following previously-described allele combinations were used: mei-41D3 (except where noted), chk2P6, spnA57/spnA093, okraAA/okraRU, spnD150, atmred31, hus137. Details about the alleles can be found at http://ﬂybase.bio.indiana.edu. The PfRr3g48C, PfRr3EJ1g48C, and TM3 Sb PfUIEg72C transgenic ﬂies were obtained from Bill Engels [30]. The following ﬂies were made by recombination: PfRr3g48C wgSP-1 L brca256E, PfRr3g48C wgSp-1 L okraRU, PfRr3EJ1g48C brca2K0, and PfRr3EJ1g48C okraAA. Antibody stainings. Ovaries were ﬁxed for 20 minutes in 1:3 4% paraformaldehyde/ PBS:heptane and other tissues were ﬁxed in 4% paraformaldehyde/PBS and blocked in 3% BSA/PBS and further processed according to standard protocols. The following modiﬁca- tions were made: the c-H2AV stainings were blocked in 10% goat serum and P-H3(Ser28) stainings were ﬁxed for 1 hour. Primary antibodies were incubated overnight at 4 8C in PBS þ 0.3% Triton (PBST) þ 1% BSA or 10% goat serum at the following concentrations: 1:500 rabbit a-c-H2AV, 1:1000 rat a-Encore, 1:500 mouse a-C(3)G clone 1A8-1G2, 1:500 rabbit a-Cleaved Caspase-3 (Cell Signaling), 1:100 mouse a-BrdU clone 3D4 (BD Biosciences), 1:500 a-P-H3(Ser28) (Upstate). 1:1000 Secondary antibodies (Molecular Probes), 1:10,000 Hoechst (Molecular Probes), or 1lg/ml Wheat-germ Agglutinin-488 (Molecular Probes) were incubated 2 hours in PBST. Repair reporter 3 assay. Details on the Rr3 assay can be found in [30–32]. Brieﬂy, individual males bearing the paternally inherited endonuclease TM3 Sb PfUIEg72C and the reporter constructs, PfRr3g48C and PfRr3EJ1g48C, were backcrossed to w1118 virgins. All matings occurred within two weeks of eclosion to minimize age- related effects. L wgSP 1 progeny were scored for eye color, presence of the endonuclease, and DsRed ﬂorescence. In cross 1, in which the EJ1 chromosome was not present, SSA repair was scored as the percent of DsRed positive non-endonuclease-bearing (endo ) prog- eny/ all endo progeny. NHEJ was scored as the percent of DsRed- negative, endonuclease-bearing (endoþ) progeny/ all endoþ progeny. In cross 2 in which both Rr3 and EJ1 were present, SSA repair was again scored as DsRed-positive endo / all endo . The DsRed-negative endoþ ﬂies represented repair by either HR-h or NHEJ and were distinguished by PCR. We used primers to speciﬁcally amplify the region containing the mutated I-SceI site on the EJ1 chromosome [30]; presence of a band indicated repair by HR-h. We also used control primers in the same PCR reaction to exclude rare unsuccessful reactions. PCR products were run on a 3% MetaPhor agarose gel (Lonza) to simultaneously distinguish between repair by short and long tract HR-h. Ten Dark endoþ progeny per male or as many as were available were used for PCR. Deletions were scored as percent of white-eyed males among total Rr3 progeny; the means and standard errors were multiplied by 2 to allow comparisons with previously reported values. The deletion class was not isolated prior to DsRed and PCR scoring and therefore also represents a small portion of the NHEJ class. Permutation tests were used to compute two-tailed P-values as described in [31]. Mutagen sensitivities. Heterozygous parents were mated and eggs were collected for 24 hours at room temperature in vials for chemical mutagenesis or apple juice plates for irradiation. 48 hours later the larvae were treated with 250 ll of 0.025% or 0.08% methyl methanesulfonate (Sigma) or irradiated with 500 or 1,250 Rads in a Faxitron X-ray cabinet. Control ﬂies were treated with 250 ll water or not irradiated. After eclosion the percent of homozygous mutant ﬂies was determined, and the sensitivity was expressed as the percent of mutant ﬂies/ total progeny in the treated vial as compared to the relative percent of mutant ﬂies in untreated control vials. Checkpoint assays. Climbing third instar larva were irradiated at 500 or 4,000 Rads in a Faxitron X-ray cabinet. For analysis of cell cycle progression and apoptosis, larva were inverted in PBS and ﬁxed after 1 and 4 hours respectively. For analysis of replication, larva were inverted in Schneider’s media 1.5 hours after irradiation. Larvae were incubated with Schneider’s media þ 10 lM BrdU (Sigma) for 30 minutes, washed 2 times 3 minutes and ﬁxed in 4% paraformalde- PLoS Genetics | www.plosgenetics.org 0010 February 2008 | Volume 4 | Issue 2 | e31