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...

During telomere replication in yeast, chromosome ends acquire an S-phase-specific overhang of the guanosine-rich strand. Here it is shown that in cells lacking Ku, a heterodimeric protein involved in nonhomologous DNA end joining, these overhangs are present throughout the cell cycle. In vivo cross-linking experiments demonstrated that Ku is bound to telomeric DNA. These results show that Ku plays a direct role in establishing a normal DNA end structure on yeast chromosomes, conceivably by functioning as a terminus-binding factor. Because Ku-mediated DNA end joining involving telomeres would result in chromosome instability, our data also suggest that Ku has a distinct function when bound to telomeres.

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

The Saccharomyces cerevisiae Ku complex, while important for nonhomologous DNA end joining, is also necessary for maintaining wild-type telomere length and a normal chromosomal DNA end structure. Yeast cells lacking Ku can grow at 23°C but are unable to do so at elevated temperatures due to an activation of DNA damage checkpoints. To gain insights into the mechanisms affected by temperature in such strains, we isolated and characterized a new allele of the YKU70 gene, yku70-30 ts . By several criteria, the Yku70-30p protein is functional at 23°C and nonfunctional at 37°C. The analyses of telomeric repeat maintenance as well as the terminal DNA end structure in strains harboring this allele alone or in strains with a combination of other mutations affecting telomere maintenance show that the altered DNA end structure in yeast cells lacking Ku is not generated in a telomerase-dependent fashion. Moreover, the single-stranded G-rich DNA on such telomeres is not detected by DNA damage checkpoints to arrest cell growth, provided that there are sufficient double-stranded telomeric repeats present. The results also demonstrate that mutations in genes negatively affecting G-strand synthesis (e.g., RIF1) or C-strand synthesis (e.g., the DNA polymerase ␣ gene) allow for the maintenance of longer telomeric repeat tracts in cells lacking Ku. Finally, extending telomeric repeat tracts in such cells at least temporarily suppresses checkpoint activation and growth defects at higher temperatures. Thus, we hypothesize that an aspect of the coordinated synthesis of double-stranded telomeric repeats is sensitive to elevated temperatures.DNA double-strand breaks (DSBs) belong to the most disruptive forms of DNA damage. If left unrepaired, they lead to broken chromosomes, which can cause cell death. On the other hand, incorrectly repaired DSBs can give rise to chromosomal aberrations such as rearrangements, deletions, and chromosome fusions (reviewed in references 17, 34, and 61). In a cell with DSBs, surveillance mechanisms called DNA damage checkpoints are activated in order to prevent DNA replication and/or cell division before the damage is repaired (79, 85). DNA damage checkpoints are likely to be responsible for activating the repair mechanisms, and they are thought to provide cells with time to complete the repair process by slowing down cell cycle progression.Telomeres are special functional complexes at the ends of eukaryotic chromosomes. Unlike DNA DSBs, the ends of eukaryotic chromosomes are stable and do not activate the DNA damage checkpoints. Thus, telomeres protect the chromosome ends from fusion and degradation and allow the cell to differentiate a chromosomal DSB from a natural end (48,52,57,70). In the vast majority of eukaryotes, including yeast and mammals, telomeres consist of simple repeated DNA sequences and their associated proteins. The strand running 5Ј to 3Ј toward the end contains clusters of three or four guanines and is commonly referred to as the G-rich strand (81). For example, in the yeast Saccharomyc...