Dm brca2 in DNA Repair and Checkpoints Figure 2. Persistent Double-Stranded Breaks in brca2 Mutants Activate the Meiotic Recombination Checkpoint (A) Egg chambers stained for DNA (green) and membranes (red). Percentage shown is the average number of wild-type-like, spherical karyosomes from two independent experiments with a total of 175–200 nuclei counted. Insert shows the region containing the oocyte nucleus. (B) Eggshells laid by wild-type and brca2KO/56 mutant females. (C) Frequency of the different classes of eggshells laid by females fed yeast for 3–4 days. Class 1 eggs have two separate dorsal appendages and appear wild-type with respect to dorsal patterning. Class 2 eggs have 2 dorsal appendages that are fused at the base. Class 3 eggs have a single dorsal appendage. Class 4 eggs have no dorsal appendages and represent the complete loss of dorsal fate. Error bars represent the standard error between 3 independent experiments. Total number of eggs counted per genotype was between 500 and 3,000 eggs. PfCG30169gi23 and PfCG30169gi48 are genomic rescue constructs inserted at two different chromosomal locations. doi:10.1371/journal.pgen.0040031.g002 Growth Factor receptor responsible for specifying dorsal fates in the Drosophila embryo and eggshell [1,34]. Activation of the meiotic recombination checkpoint results in the ventralization of eggs laid by females mutant for meiotic DSB repair genes. A second characteristic phenotype of checkpoint activation is the failure to properly form the karyosome of the oocyte nucleus. These defects can be suppressed by mutations in the kinases required for checkpoint transduction indicating that they are due to checkpoint activity [1]. Egg chambers from brca2 mutant females had a highly penetrant karyosome defect (Figure 2A). In addition, brca2 mutant females laid weakly ventralized eggs that did not hatch (Figure 2B and 2C). The eggshell patterning and karyosome formation defects can be rescued by a single copy of a genomic rescue construct (Figure 2A and 2C). Transduction of the meiotic recombination checkpoint signal requires Hus1, a member of the Rad9-Hus1-Rad1 checkpoint complex (9-1-1), and the checkpoint kinases Mei- 41/ATR and Chk2 [1,35,36]. Mutations in either mei-P22, a gene required for DSB formation [37], or the checkpoint kinase chk2 suppress the eggshell and karyosome defects of brca2 mutants (Figure 2A and 2C), clearly demonstrating that the ovarian phenotypes observed are due to persistent DSBs that activate the meiotic checkpoint. We observed little or no suppression of either the eggshell or the karyosome defects in mei41D3; brca2KO/56 (Figure 2C) or mei4129D; brca2KO/56 (data not shown) double mutants. This may be due to a possible redundancy between the two upstream checkpoint kinases Mei-41/ATR and ATM. Currently it is not possible to test for a role of atm in the meiotic recombination checkpoint as even the hypomorphic, viable atm mutants available have eggshell patterning and karyosome defects ([38] and our unpublished results). Additional explanations for the lack of mei-41 suppression of the brca2 ovarian phenotype are presented in the Discussion. Nevertheless, suppression by chk2 muta- PLoS Genetics | www.plosgenetics.org 0005 February 2008 | Volume 4 | Issue 2 | e31