RPA initially binds to the 3′ overhangs and is subsequently replaced by Rad51 to promote homologous pairing and strand invasion ( 5).
HR initiates by nucleolytic degradation of DNA ends to generate long 3′ single-strand DNA (ssDNA) tails, a process termed end resection. HR employs extensive homology and templated DNA synthesis to restore the broken chromosome and is considered to be a mostly error-free mode of repair ( 4). The Ku heterodimer (Ku70-Ku80), an essential NHEJ component, binds to DSB ends to protect them from degradation and recruits the DNA ligase IV complex to catalyze end ligation ( 3). Typically, cells repair DSBs by nonhomologous end joining (NHEJ) or by homologous recombination (HR) ( 2). DNA double-strand breaks (DSBs) are one of the most cytotoxic forms of DNA damage and can cause loss of genetic information, gross chromosome rearrangements, or even cell death in the absence of the appropriate response. Cells respond to these insults by an elaborate network of surveillance mechanisms and DNA repair pathways, referred to as the DNA damage response (DDR) ( 1). Genomic integrity is constantly threatened by DNA damage that can result from exposure to exogenous sources, such as ionizing radiation, as well as from endogenous sources, including DNA replication errors and intermediates in excision repair or topoisomerase transactions. Collectively, these data show a resection initiation independent role for Sae2 at DSBs by modulating the DNA damage checkpoint. We suggest that persistent Sae2 binding at DSBs in the mre11-nd mutant counteracts the inhibitory effects of Rad9 and Rad53 on Exo1 and Dna2-Sgs1–mediated resection, accounting for the different phenotypes conferred by mre11-nd and sae2Δ mutations. We show that Sae2 competes with other Tel1 substrates, thus reducing Rad9 binding to chromatin and to Rad53. Third, Sae2 attenuates the Tel1-Rad53 CHK2 checkpoint and antagonizes Rad9 53BP1 accumulation at DSBs independent of Mre11 nuclease. Second, sae2Δ is synthetic lethal with sgs1Δ, whereas the mre11-nd sgs1Δ mutant is viable. First, sae2Δ cells exhibit greater sensitivity to genotoxins than mre11-nd cells. Accordingly, Mre11-nuclease deficient ( mre11-nd) and sae2Δ mutants are expected to exhibit similar phenotypes however, we found several notable differences. To initiate resection, Mre11 endonuclease nicks the 5′ strands at DSB ends in a reaction stimulated by Sae2 CtIP. The Mre11-Rad50-Xrs2 NBS1 complex plays important roles in the DNA damage response by activating the Tel1 ATM kinase and catalyzing 5′–3′ resection at DNA double-strand breaks (DSBs).
0 kommentar(er)
