Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double-strand break (DSB)-induced extensive LOH, and its suppression, using a non-essential minichromosome, Ch 16 , in fission yeast. We find extensive LOH to arise from a new break-induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1-dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break-induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genomewide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.

Nucleotide synthesis is a universal response to DNA damage, but how this response facilitates DNA repair and cell survival is unclear. Here we establish a role for DNA damage-induced nucleotide synthesis in homologous recombination (HR) repair in fission yeast. Using a genetic screen, we found the Ddb1-Cul4 Cdt2 ubiquitin ligase complex and ribonucleotide reductase (RNR) to be required for HR repair of a DNA double-strand break (DSB). The Ddb1-Cul4 Cdt2 ubiquitin ligase complex is required for degradation of Spd1, an inhibitor of RNR in fission yeast. Accordingly, deleting spd1 + suppressed the DNA damage sensitivity and the reduced HR efficiency associated with loss of ddb1 + or cdt2 + . Furthermore, we demonstrate a role for nucleotide synthesis in postsynaptic gap filling of resected ssDNA ends during HR repair. Finally, we define a role for Rad3 (ATR) in nucleotide synthesis and HR through increasing Cdt2 nuclear levels in response to DNA damage. Our findings support a model in which breakinduced Rad3 and Ddb1-Cul4 Cdt2 ubiquitin ligase-dependent Spd1 degradation and RNR activation promotes postsynaptic ssDNA gap filling during HR repair.[Keywords: Rad3; Ddb1-Cul4 Cdt2 ubiquitin ligase; Spd1; ribonucleotide reductase; homologous recombination repair; fission yeast] Supplemental material is available at http://www.genesdev.org. Received July 16, 2010; revised version accepted October 15, 2010. DNA double-strand breaks (DSBs) are potentially lethal lesions that, if left undetected or repaired incorrectly, can threaten the integrity of the genome. DSBs arise at a low frequency during normal cell metabolism and can also arise from exposure to DNA-damaging agents such as ionizing radiation (IR) (Shrivastav et al. 2008), potentially leading to chromosomal rearrangements, cancer, or cell death (Pfeiffer et al. 2000). Consequently, an intricate network of cellular responses for detection and accurate repair of such lesions exists within the cell (Jackson and Bartek 2009).Cells have evolved two distinct repair pathways to maintain genome integrity following a DSB: nonhomologous end-joining (NHEJ), in which DNA ends are directly ligated, and homologous recombination (HR) (Shrivastav et al. 2008). In yeast, HR involves the RAD52 epistasis group (Krogh and Symington 2004) and uses a homologous sequence as a template for repair-typically the sister chromatid or, less frequently, the homologous chromosome (Kadyk and Hartwell 1992). Repair is initiated by 59-39 resection of the broken ends to form a 39 ssDNA overhang. This is a two-step process in which MRX/MRN (Mre11-Rad50-Xrs2 in Saccharomyces cerevisiae (Sung 1997;Sugawara et al. 2003;Wolner et al. 2003;Haruta et al. 2008). Stabilization of the nucleoprotein filament is achieved through interaction between Rad51 and Rad54; strand invasion is then initiated, resulting in the formation of a displacement (D) loop (Petukhova et al. 1998;Mazin et al. 2003;Sugawara et al. 2003). RPA further functions postsynaptically to stabilize DNA pairing and possibly the displaced ssD...

DNA double-strand breaks (DSBs) can cause chromosomal rearrangements and extensive loss of heterozygosity (LOH), hallmarks of cancer cells. Yet, how such events are normally suppressed is unclear. Here we identify roles for the DNA damage checkpoint pathway in facilitating homologous recombination (HR) repair and suppressing extensive LOH and chromosomal rearrangements in response to a DSB. Accordingly, deletion of Rad3ATR, Rad26ATRIP, Crb253BP1 or Cdc25 overexpression leads to reduced HR and increased break-induced chromosome loss and rearrangements. We find the DNA damage checkpoint pathway facilitates HR, in part, by promoting break-induced Cdt2-dependent nucleotide synthesis. We also identify additional roles for Rad17, the 9-1-1 complex and Chk1 activation in facilitating break-induced extensive resection and chromosome loss, thereby suppressing extensive LOH. Loss of Rad17 or the 9-1-1 complex results in a striking increase in break-induced isochromosome formation and very low levels of chromosome loss, suggesting the 9-1-1 complex acts as a nuclease processivity factor to facilitate extensive resection. Further, our data suggest redundant roles for Rad3ATR and Exo1 in facilitating extensive resection. We propose that the DNA damage checkpoint pathway coordinates resection and nucleotide synthesis, thereby promoting efficient HR repair and genome stability.