Dm brca2 in DNA Repair and Checkpoints Figure 3. brca2 Mutants Suppress the Patterning Defects of DNA Repair Mutants That Activate the Meiotic Recombination Checkpoint (A) c-H2AV(red) foci in the germarium, with the oocyte in region 3 marked by the arrow. Pro-oocytes are marked by C(3)G staining (blue). Images represent a projection of stacks taken through whole germaria. Number of c-H2AV foci in region 3 is the average number of foci þ/ standard error from a total of 14, 14, 11, 7, 10, and 10 region 3 oocytes counted for OreR, brca2, spnA, okra, brca2; spnA, and brca2 okra, respectively. (B) Frequency of the different classes of eggshells laid by females fed yeast for 5–7 days. Classes are as described in Figure 2. Note the increase in the wild-type like eggshells (blue) in the double mutants. Error bars represent the standard error between 3 independent experiments. Total number of eggs counted per genotype were: 1149, 73, 1687, 456, 645, 1663, 922, 1199, 341, 339, 455, 268, and 196, respectively. doi:10.1371/journal.pgen.0040031.g003 tions indicate that the ovarian phenotypes of brca2 mutants result from checkpoint activation. Brca2 Is Also Required for Efficient Activation of the Meiotic Recombination Checkpoint The brca2 eggshell patterning defect is a considerably weaker defect than that of any of the meiotic DNA repair mutants known to activate the checkpoint, spnA/rad51, spnB/ xrcc3, spnC/hel308, spnD/rad51C, or okra/rad54 [14,39–41]. We examined the levels of c-H2AV, a marker for DSBs, in order to determine if the number of DSBs was reduced in brca2 mutants relative to other meiotic DSB repair mutants, or ‘‘spindle class’’ genes. In wild-type ﬂies c-H2AV foci are seen in pro-oocyte nuclei in regions 2a and occasionally 2b and are absent from region 3 [42]. An average of 14–15 c-H2AV foci are present in a pro-oocyte nucleus in region 2a before declining in region 2b. Meiotic DNA repair mutants show a delay in c-H2AV foci formation, but reach a peak number of 20–24 c-H2AV foci in region 3 [42]. brca2 mutants had 19.2 c- H2AV foci as compared to 21.1 foci in spnA mutants in region 3 (Figure 3A). Therefore it is unlikely that the weak phenotype seen in brca2 mutants is due to decreased accumulation of persistent DSBs. Surprisingly, when we made double mutants with brca2 and any of the other meiotic DNA repair mutants, brca2 strongly suppressed the eggshell phenotype. For example, spnD mutant females lay 79.3% strongly ventralized eggs (class3 and class 4 eggs) and 13.8% wild-type-like eggs (class1). brca2; spnD mutant females lay 18.7% strongly ventralized eggs and 79.3% wild-type like eggs. Similar results were seen with okra and spnA mutants (Figure 3B). In order to determine if the suppression of the eggshell phenotype was due to increased DSB repair in the double mutants, we examined the number of c-H2AV foci present in the double mutants. spnA, okra, and the double mutants with brca2 all had similar levels of c-H2AV foci in region 3 of the germarium (Figure 3A). Therefore it is unlikely that increased levels of repair occur in the double mutants, and we propose that Brca2 has a second role in meiosis in transducing the meiotic recombination checkpoint signal. brca2 and hus1 Are Not Required for Checkpoints That Respond to Irradiation Damage The DNA damage checkpoints that respond to multiple types of damage and result in diverse damage responses converge upon a common set of related transducer kinases PLoS Genetics | www.plosgenetics.org 0006 February 2008 | Volume 4 | Issue 2 | e31