Break-induced ATR and Ddb1–Cul4^Cdt2 ubiquitin ligase-dependent nucleotide synthesis promotes homologous recombination repair in fission yeast

Abstract

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...

A Mammalian-Like DNA Damage Response of Fission Yeast to Nucleoside Analogs

“…Alternatively, transient changes in one dNTP during spd1Δ replication may not prompt arrest. Our data are consistent with models linking dNTP regulation to genome stability in S. pombe (Holmberg et al 2005;Moss et al 2010;Nestoras et al 2010). While chk1Δ hsv-tk + hENT + cells do not show increased mutation post-BrdU, we hypothesize that a spd1Δ chk1Δ double mutant may have a very high mutation rate due to extra damage and larger dNTP pools.…”

“…Spd1 is ubiquitinated during stress so that the dNTP pool is expanded, which ties nucleotide metabolism to the cell cycle and DDR (Moss et al 2010;Nestoras et al 2010). We found that spd1Δ cells are less sensitive to chronic nucleoside exposure, but their relative viability in acute BrdU is worse.…”

Spd1 accumulation causes genome instability independently of ribonucleotide reduction but functions to protect the genome when deoxynucleotide pools are elevated

“…The CRL4 complex is also present in the fission yeast Schizosaccharomyces pombe, but not in the distantly related budding yeast Saccharomyces cerevisiae. In S. pombe, mutations that abrogate the function of CRL4 Cdt2 cause increased mutation rates, DNA damage sensitivity, slow progression through S phase, activation of and dependency on the DNA damage and integrity checkpoints, and failure to proceed through premeiotic S phase (Liu et al, 2003;Bondar et al, 2004;Holmberg et al, 2005;Liu et al, 2005;Moss et al, 2010). These parts of the phenotype can be suppressed to varying extents by simultaneous deletion of spd1 , which encodes a small intrinsically disordered protein (Nestoras et al, 2010).…”

Cofactor Strap regulates oxidative phosphorylation and mitochondrial p53 activity through ATP synthase

“…The total ATP levels were measured as described previously 40 with slight modifications. Cells were seeded in 10-cm dishes.…”

“…The aqueous upper layer was removed, and 5-10 ml was injected for HPLC analysis as described previously. 40 ATP was identified and quantitated against commercially available ATP.…”