DNA helicases Sgs1 and BLM promote DNA double-strand break resection

Abstract

A key cellular response to DNA double-strand breaks (DSBs) is 5-to-3 DSB resection by nucleases to generate regions of ssDNA that then trigger cell cycle checkpoint signaling and DSB repair by homologous recombination (HR). Here, we reveal that in the absence of exonuclease Exo1 activity, deletion or mutation of the Saccharomyces cerevisiae RecQ-family helicase, Sgs1, causes pronounced hypersensitivity to DSB-inducing agents. Moreover, we establish that this reflects severely compromised DSB resection, deficient DNA damage signaling, and strongly impaired HR-mediated repair. Furthermore, we show that the mammalian Sgs1 ortholog, BLMwhose deficiency causes cancer predisposition and infertility in people-also functions in parallel with Exo1 to promote DSB resection, DSB signaling and resistance to DSB-generating agents. Collectively, these data establish evolutionarily conserved roles for the BLM and Sgs1 helicases in DSB processing, signaling, and repair. Results and DiscussionDNA double-strand breaks (DSBs) are highly cytotoxic lesions that are induced by ionizing radiation (IR) and certain anti-cancer chemotherapeutic agents. To survive DSB exposure and maximize genome stability, cells possess a complex machinery to detect DSBs, signal their presence, and mediate their repair (Zhou and Elledge 2000;Rouse and Jackson 2002;Lisby and Rothstein 2004;Wyman and Kanaar 2006). The importance of such responses is revealed by the fact that inherited defects in them lead to human pathologies, including cancer, infertility and neurodegeneration (Khanna and Jackson 2001;Kastan and Bartek 2004). In all eukaryotes, an early response to DSBs is their 5Ј-to-3Ј resection to produce ssDNA (Lisby and Rothstein 2004;Wyman and Kanaar 2006). This is bound by replication protein A (RPA), which then recruits and activates the human protein kinase ATR (Mec1 in Saccharomyces cerevisiae) to phosphorylate downstream effector proteins, including the protein kinase CHK1 (the functional counterpart of S. cerevisiae Rad53;Zou and Elledge 2003). In addition to promoting signaling responses, ssDNA regions are also required for DNA repair by homologous recombination (HR), being bound by the HR proteins Rad51 and Rad52 (Wyman and Kanaar 2006;San Filippo et al. 2008). To understand the mechanisms and control of DSB signaling and HR repair, it is therefore crucial to define how DSBs are detected and processed into ssDNA.While the S. cerevisiae nuclease Exo1 promotes DSB resection, its contribution to this is modest, with exo1⌬ mutants exhibiting considerable residual resection and displaying little hypersensitivity to DNA damaging agents (Moreau et al. 2001;Maringele and Lydall 2002;Nakada et al. 2004;Tran et al. 2004;Cotta-Ramusino et al. 2005;Clerici et al. 2006;Bermejo et al. 2007). Furthermore, yeast cells deficient in the Mre11-Rad50-Xrs2 (MRX) complex also display impaired DSB processing (Lee et al. 1998;Nakada et al. 2004;Clerici et al. 2006), with the resection defect of mrx⌬ exo1⌬ double mutants being more severe than those of the single m...

Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells

“…We found that whereas the Mre11 complex and CtIP are required for the initial short-range end resection to promote MMEJ, BLM and Exo1 are dispensable for this process but are needed for extended end resection and HR (Fig. 5C) (36,43), thus revealing different genetic requirements for the two distinct end resection steps in mammalian cells. Notably, the need for Mre11 nuclease activity in end resection and HR is different in yeast and mammals.…”

Differential contribution of HP1 proteins to DNA end resection and homology-directed repair

“…Given that CtIP recruitment is not affected after HP1 knockdown (Fig. 2E), to investigate potential links with other relevant proteins that work downstream CtIP, like the Bloom helicase (BLM) and the exonuclease EXO1, 26,27 will be therefore critical to clarify the role of HP1 proteins during DNA-end resection.…”

Yeast Mph1 helicase dissociates Rad51-made D-loops: implications for crossover control in mitotic recombination

“…The Sgs1 helicase plays distinct roles in DSB end resection (Gravel et al 2008;Mimitou and Symington 2008;Zhu et al 2008), the DNA replication checkpoint (Frei and Gasser 2000), the control of homeologous recombination (Myung et al 2001), and in Figure 7. Mechanism and specificity of Mph1 action.…”