Author Summary Breast cancer can arise due to inherited mutations in a few well- defined breast cancer susceptibility genes. BRCA2 is one of two known human genes in which common mutations are associated with high breast cancer risk. A known function of BRCA2 is the repair of damaged DNA using the homologous recombination repair pathway. In the absence of BRCA2, damaged DNA is repaired by error-prone repair. However, as many other genes required for homologous recombination are not known breast cancer suscept- ibility genes, BRCA2 likely has additional roles in tumor suppression. In this study, we created fruit flies mutant for the homolog of BRCA2. We show that, like in humans, fruit fly Brca2 is required for DNA repair by homologous recombination. Additionally, we use this new animal model to discover a previously unknown role for Brca2 in the production of a signal that tells cells that damaged DNA is present during meiosis. This ‘‘checkpoint’’ signal may allow the cells to coordinate a protective response after DNA damage. checkpoint stabilizes replication forks and decreases repli- cation levels in response to lesions at the replication fork. Requirements for BRCA2 in replication fork stabilization after hydroxyurea treatment and in suppressing radioresist- ant replication have also been described [9,10]. The G1/S, S/M, and G2/M checkpoints have been found to be largely intact in BRCA2 mutants. However, these studies have used hypomor- phic mutations that preserve half or more of the N-terminal region of BRCA2 due to proliferative defects of BRCA2 mutants [3,10]. As modest to severe increases in breast cancer susceptibility have been associated with mutations in other checkpoint genes [11], further elucidation of the mechanism of the role of BRCA2 in checkpoints is needed. Drosophila has emerged as a useful model for studying DNA repair and checkpoint control of genome stability. The DNA repair and checkpoint pathways are remarkably well conserved between ﬂies and higher organisms [12]. Notably, though the function of many of these genes is well conserved, mutants in Drosophila are sometimes viable in cases in which mutations in higher organisms result in lethality and complicate mammalian developmental studies [13,14]. We made mutations in the Drosophila homolog of BRCA2 (CG30169), and show that unlike in mammals, Drosophila brca2 mutants are viable. We present detailed descrip- tions of DSB repair pathway balance and irradiation-induced checkpoint function in animals genetically for brca2. CG30169 represents a functional BRCA2 homolog required for DSB repair in mitotic and meiotic tissues. Additionally, we uncover a novel role for brca2 in the meiotic recombination checkpoint. Finally we show that Brca2 co-immunoprecipi- tates with the checkpoint protein Rad9, suggesting a mechanism for the role of Brca2 in checkpoint control. Results Null Mutations in the Predicted Drosophila BRCA2 Homolog, CG30169, Are Viable Poor sequence conservation of BRCA2 homologs outside of the BRC repeats and DNA binding domains initially hampered the identiﬁcation and study of BRCA2 in lower model organisms. However, by using a sequence motif that describes the BRC repeats, Lo et al. (2003) identiﬁed putative homologs across many species [15], including some that have since been veriﬁed to be functional homologs [16,17]. One Dm brca2 in DNA Repair and Checkpoints gene in Drosophila, CG30169, was predicted to encode BRC repeats. CG30169 encodes a 109 kDa protein containing 3 putative BRC repeats and 4 Nuclear Localization Sequences. Notably, while Lo et al. (2003) predicted DNA binding domains in vertebrate, plant, and some lower eukaryotes, they did not ﬁnd evidence of a DNA binding domain in CG30169 by sequence analysis. Therefore it was not self- evident that CG30169 encodes a functional BRCA2 homolog, since the expression of isolated repeats is known to dominantly inhibit Rad51-dependent DSB repair, but fusions of a BRC repeat to Replication Protein A restore DNA binding and are consequently able to rescue the repair defects of BRCA2 mutants [18–20]. In order to determine if CG30169 encodes a functional BRCA2 homolog and examine its role in the DNA damage responses, we made mutations in CG30169. brca256E was created by imprecise excision of the P element PfSUPor- P CG30169KG03961 which is inserted in the 5’UTR of CG30169. g brca256E removes the majority of the coding sequence of CG30169 and a portion of the 5’UTR of the neighboring gene CG4612. brca2KO was created by ends-out homologous recombination [21], and the resulting chromosome is completely deleted for CG30169 but contains fully intact neighboring genes. We found that homozygous brca2KO, brca256E, and transheterozygous mutant combinations are viable but recessive female sterile. The Drosophila BRCA2 Homolog Is Required for Homologous Recombination Repair The best characterized role of BRCA2 is in the regulation of the recombinase Rad51, which has been shown to be required for DNA repair in Drosophila [14]. In order to determine if CG30169 represented a functional BRCA2 homolog we ﬁrst tested for a role in somatic DNA repair by assaying sensitivities to the mutagens, methyl methanesulfonate (MMS) and x-rays. X-ray irradiation (IR) results in a range of DNA damage including a large number of DSBs through- out the cell cycle, while MMS leads to replication fork collapse and DSB formation speciﬁcally during S phase. We found that similar to rad51/spnA and rad54/okra mutants ([14] and Tables 1 and 2), brca2 mutants are highly sensitive to both MMS and IR. Flies heterozygous for either brca2 allele were crossed together, the larval progeny were exposed to mutagen, and the percent of homozygous mutant ﬂies that survive to adulthood among all surviving adults was determined. brca2 mutants showed complete to near-complete lethality after a 1250 Rad IR exposure and 3–13-fold decrease in survivorship after exposure to 500 Rads of irradiation (Table 1). Exposure to 0.08% MMS resulted in a 5–24-fold decrease in survivor- ship of brca2 mutants (Table 2). Flies doubly mutant for okra and brca2 showed mutagen sensitivities comparable to the single mutants alone, suggesting the two genes act in the same pathway. The similar mutagen sensitivities between CG30169, okra, and spnA mutants suggest that CG30169 may be a functional BRCA2 homolog involved in DSB repair. Several pathways are responsible for the repair of DSBs and the choice between these pathways is a highly regulated process inﬂuenced by factors such as the nature of the break or the phase of the cell cycle [22,23]. Three major pathways known to repair DSBs are: non-homologous end-joining (NHEJ), homologous recombination (HR), and single-strand annealing (SSA) [4]. In NHEJ, the DNA ends ﬂanking the PLoS Genetics | www.plosgenetics.org 0002 February 2008 | Volume 4 | Issue 2 | e31