Dm brca2 in DNA Repair and Checkpoints Table 1. Irradiation Sensitivity of brca2 Mutants Table 2. Methyl Methanesulfonate Sensitivity of brca2 Mutants Genotype 0 Rads 500 Rads 1250 Rads n ¼ Genotype 0% MMS 0.025% MMS 0.08% MMS n ¼ brca2KO/56 35.5 (3.4) brca2KO 28.6 (2.1) brca2KO/þ a 49.1 (0.9) okraAA/RU 33.7 (0.6) brca2KO/56okraAA/RU 34.0 (1.7) 2.7 (0.9) 10.1 (2.1) 48.1 (2.2) 9.3 (2.3) 3.1 (1.0) 0 (0) 0.3 (0.3) 52.2 (1.0) 0 (0) 0 (0) 540, 295, 676 2215, 1893, 1537 2757, 2156, 2720 1495, 616, 1339 2105, 1521, 598 Percent of homozygous mutant progeny among all surviving progeny. Heterozygous parents were mated and the surviving adult progeny were counted. All mutant chromosomes were balanced over CyO. Standard errors between experiments are indicated in parentheses and n indicates the total number of progeny counted. The mutant progeny were expected to constitute 33.3% of the progeny except for the brca2KO/þ progeny which were expected to comprise 50.0% of the progeny. aTo measure any dominant effects of the brca2 chromosome, brca2KO /CyO flies were backcrossed to OreR and the percentage of brca2KO/þ flies was measured. doi:10.1371/journal.pgen.0040031.t001 break are annealed, resulting in the insertion or deletion of several nucleotides at the site of the break. During SSA repair stretches of repetitive DNA ﬂanking the DSB are annealed. Due to the highly repetitive nature of eukaryotic chromo- somes this pathway is likely available to some, but not all, naturally occurring DSBs. HR uses homologous sequences on the homologous chromosome or sister chromatid as a template for repair. If the sister chromatid is used, HR is non-mutagenic. However, if the homologous chromosome is the template, loss of heterozygosity can occur. BRCA2 in other species has been shown to be required for Rad51- dependent repair by homologous recombination and other roles in DNA repair have been suggested [16,17,24]. In C. elegans there is in vitro and indirect in vivo evidence for a role for the C. elegans BRCA2 homolog in SSA [16,25], though in mammalian cell culture studies with hypomorphic mutations, an increase in SSA was observed [26–29]. We used the Repair Reporter 3 (Rr3) assay [30] to simultaneously measure the relative levels of HR, NHEJ, and SSA repair in the pre-meiotic germline of individual males (Figure 1). The Rr3 assay monitors the repair of a DSB at an I- SceI endonuclease site ﬂanked by partial copies of the reporter dsRed. There is a 147 base pair (bp) tandem duplication of dsRed ﬂanking the break. I-SceI is ubiquitously expressed at the onset of zygotic transcription and the repair outcomes of DSBs in the male germline can be observed in the progeny (Figure 1A). Repair by SSA recreates a functional copy of dsRed and the resulting ﬂies are ﬂuorescent red even in the absence of the endonuclease. Repair by NHEJ creates small insertions or deletions at the I-SceI site and the resulting ﬂies are dark even in the presence of endonuclease. These two classes were scored from populations either with or without endonuclease in order to distinguish them from the uncut reporter carrying ﬂies, which are mosaic red and often undistinguishable from germline DsRed ﬂies [30]. Repair by HR using the sister chromatid (HR-s) recreates the reporter, which can be re-cut until a terminal outcome is reached. HR repair using the homologous chromosome (HR- h) can occur when a Rr3 variant, EJ1, which contains a mutated I-SceI site is present on the homologous chromo- some. Repair by HR-h results in dark ﬂies even when the endonuclease is present and can be distinguished from NHEJ by PCR speciﬁc to the mutated I-SceI site. brca2KO/56 37.3 (2.7) brca2KO 30.8 (2.3) brca2KO/þ a 49.6 (1.9) okraAA/RU 30.1 (2.2) brca2KO/56okraAA/RU 33.3 (3.1) spnA093/57 37.3 (2.7) 37.6 (1.6) 32.5 (0.9) 49.1 (2.3) 32.3 (1.6) 31.3 (1.5) 37.4 (2.3) 7.4 (1.5) 1.3 (0.5) 48.8 (1.6) 4.3 (1.6) 1.2 (0.7) 14.2 (1.5) 405, 685, 302 965, 1405, 864 1135, 808, 968 942, 1143, 728 521, 973, 399 405, 991, 918 Percent of homozygous mutant progeny among all surviving progeny. Heterozygous parents were mated and the surviving adult progeny were counted. brca2 and okra were balanced over CyO and spnA was balanced over TM3. Standard errors between experiments are indicated in parentheses and n indicates the total number of progeny counted. The mutant progeny were expected to constitute 33.3% of the progeny except for the brca2KO/þ progeny which were expected to comprise 50.0% of the progeny. aTo measure any dominant effects of the brca2 chromosome, brca2KO /CyO flies were backcrossed to OreR and the percentage of brca2KO/þ flies was measured. doi:10.1371/journal.pgen.0040031.t002 We measured two additional parameters of DSB repair. First, we monitored the extent of gene conversion to the left of the break by determining if the repaired chromosome included a 16 bp deletion 156 bp away from the DSB that was speciﬁc to the parental EJ1 chromosome. We designate repaired chromosomes containing the 16 bp deletion as being repaired by long-tract HR-h. Long-tract HR-h repair likely represents repair in which there is increased gene conversion due to increased DNA synthesis rates following strand invasion or due to increased resection prior to DNA synthesis. Alternatively, changes in the repair of the hetero- duplex DNA could also inﬂuence the balance of repair products observed. Second, large deletions to the left of the DSB result in progeny with white eyes due to the loss of the white gene carried on the Rr3 transgene. We performed the Rr3 assay with and without the EJ1 chromosome in order to measure the repair products when repair by HR-h is available and unavailable. All hypothesis testing was done using P- values derived from permutation tests described in [31], which account for potential clustering of outcomes in single males due to multiple sperm produced from a single repair event. In cross 2, in which the EJ1 chromosome was present and the HR-h pathway was available, we saw highly signiﬁcant decreases in HR-h repair in brca2 mutants (P ,1e 8) (Figure 1B). Short-tract HR-h was decreased 64-fold from wild-type values and long-tract HR-h was decreased 9-fold. These values likely underestimate the requirement for brca2 in HR-h. Due to the female sterility of brca2, repair pathway use was measured in the genetically-null offspring of females hetero- zygous for brca2 and low levels of Brca2 protein may still have been present at the onset of the zygotic transcription when the endonuclease was initially expressed. We also observed a 5-fold increase in ﬂanking deletions in brca2 mutants. We did not see a signiﬁcant increase in deletions in cross 1 in which HR-h was not available (Figure 1C). Therefore, the deletions most likely resulted from aberrant HR-h repair, as has been previously suggested for spnA and okra mutants [32]. We did not observe a requirement for brca2 in SSA. In fact, there was a signiﬁcant increase in SSA in brca2 mutants in both cross 1 and 2 (P ¼1.4e 7 and P ,1.0e 8 respectively, Figure 1B,C). We observed signiﬁcant changes in SSA and NHEJ in cross 1 when HR-h was unavailable (P ¼1.4 e 7 and P ¼4.3 e 4) PLoS Genetics | www.plosgenetics.org 0003 February 2008 | Volume 4 | Issue 2 | e31