Drosophila brca2 Is Required for Mitotic and Meiotic DNA Repair and Efficient Activation of the Meiotic Recombination Checkpoint Martha Klovstad, Uri Abdu¤, Trudi Schu¨ pbach* Howard Hughes Medical Institute, Department of Molecular Biology, Princeton University, Princeton, New Jersey, United States of America Heterozygous mutations in the tumor suppressor BRCA2 confer a high risk of breast and other cancers in humans. BRCA2 maintains genome stability in part through the regulation of Rad51-dependent homologous recombination. Much about its precise function in the DNA damage responses is, however, not yet known. We have made mutations in the Drosophila homolog of BRCA2 and measured the levels of homologous recombination, non- homologous end-joining, and single-strand annealing in the pre-meiotic germline of Drosophila males. We show that repair by homologous recombination is dramatically decreased in Drosophila brca2 mutants. Instead, large flanking deletions are formed, and repair by the non-conservative single-strand annealing pathway predominates. We further show that during meiosis, Drosophila Brca2 has a dual role in the repair of meiotic double-stranded breaks and the efficient activation of the meiotic recombination checkpoint. The eggshell patterning defects that result from activation of the meiotic recombination checkpoint in other meiotic DNA repair mutants can be strongly suppressed by mutations in brca2. In addition, Brca2 co-immunoprecipitates with the checkpoint protein Rad9, suggesting a direct role for Brca2 in the transduction of the meiotic recombination checkpoint signal. Citation: Klovstad M, Abdu U, Schu¨ pbach T (2008) Drosophila brca2 is required for mitotic and meiotic DNA repair and efficient activation of the meiotic recombination checkpoint. PLoS Genet 4(2): e31. doi:10.1371/journal.pgen.0040031 Introduction The genomic stability of eukaryotic cells is constantly challenged by exogenous and endogenous stresses that can lead to the loss or alteration of genetic material. Genomic stability is maintained through robust DNA repair and checkpoint pathways that are tightly coordinated with each other and the developmental cell cycle progression of the organism. For example, mutations in meiotic DNA repair enzymes in Drosophila cause defects in the cell cycle and developmental progression of the egg due to a failure to repair meiotic recombination intermediates [1]. The tumor suppressor and breast cancer susceptibility gene, BRCA2, has been implicated in playing a central role in maintaining genomic stability, but the extent to which BRCA2 is involved the coordination of DNA repair, checkpoints, and devel- opmental progression remains to be determined. Murine cells depleted for BRCA2 spontaneously accumu- late broken chromosomes and chromatids, triradial and quadriradial structures, and gross chromosomal rearrange- ments [2,3]. A key function of BRCA2 is the regulation of the Rad51 recombinase during DNA repair by homologous recombination (HR). During HR, Rad51 assembles into a nucleoprotein ﬁlament with single-stranded DNA at the site of a double-stranded break (DSB) in order to initiate strand invasion of the homologous chromosome [4]. Recent struc- tural studies have illuminated how BRCA2 regulates Rad51 [5–7]. BRCA2 contains two regions that mediate binding to Rad51: a stretch of 8 repeated short motifs termed the BRC repeats and a C-terminal region termed TR2. The BRC repeats bind the Rad51 oligermerization domain to disrupt Rad51 self-oligermerization. BRCA2 then catalyzes the for- mation of the nucleoprotein ﬁlament at the single-stranded/ double-stranded DNA junction ﬂanking a DSB. This ﬁlament is stabilized in a cell-cycle–dependent manner by the TR2 domain of BRCA2. The role of BRCA2 in homologous recombination is likely critical for its role as a tumor suppressor, but BRCA2 is a large protein with many binding partners and it is likely to play multiple roles in safeguarding genomic stability. Several requirements for BRCA2 outside of homologous recombination have been suggested by protein interaction and cell culture studies, but these functions are far less understood. Most notably, BRCA2 has been implicated in two S-phase checkpoints: the intra-S phase checkpoint and the replication checkpoint. During the intra-S phase checkpoint irradiation-induced lesions outside of the replication fork cause partial depression of replication [8]. The replication Editor: R. Scott Hawley, Stowers Institute for Medical Research, United States of America Received October 2, 2007; Accepted December 21, 2007; Published February 8, 2008 A previous version of this article appeared as an Early Online Release on January 8, 2008 (doi:10.1371/journal.pgen.0040031.eor). Copyright: 2008 Klovstad et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abbreviations: 9-1-1, Rad9-Rad1-Hus1 complex; bp, base pair; BrdU, 5-bromo-2- deoxyuridine; DSB, double-stranded break; HR, homologous recombination; HR-h, homologous recombination using the homologous chromosome; HR-s, homolo- gous recombination using the sister chromatid; IR, irradiation; MMS, methyl methanesulfonate; NHEJ, non-homologous end joining; Rr3, Repair reporter 3; SSA, single-strand annealing * To whom correspondence should be addressed. E-mail: schupbac@princeton.edu ¤ Current address: Department of Life Sciences and the National Institute for Biotechnology in the Negev, Ben-Gurion University, Beer-Sheva, Israel PLoS Genetics | www.plosgenetics.org 0001 February 2008 | Volume 4 | Issue 2 | e31