Resection Activity of the Sgs1 Helicase Alters the Affinity of DNA Ends for Homologous Recombination Proteins inSaccharomyces cerevisiae

Abstract

The RecQ helicase family is critical during DNA damage repair, and mutations in these proteins are associated with Bloom, Werner, or Rothmund-Thompson syndromes in humans, leading to cancer predisposition and/or premature aging. In the budding yeast Saccharomyces cerevisiae, mutations in the RecQ homolog, SGS1, phenocopy many of the defects observed in the human syndromes. One challenge to studying RecQ helicases is that their disruption leads to a pleiotropic phenotype. Using yeast, we show that the separation-of-function allele of SGS1, sgs1-D664Δ, has impaired activity at DNA ends, resulting in a resection processivity defect. Compromising Sgs1 resection function in the absence of the Sae2 nuclease causes slow growth, which is alleviated by making the DNA ends accessible to Exo1 nuclease. Furthermore, fluorescent microscopy studies reveal that, when Sgs1 resection activity is compromised in sae2Δ cells, Mre11 repair foci persist. We suggest a model where the role of Sgs1 in end resection along with Sae2 is important for removing Mre11 from DNA ends during repair. REPAIR of DNA damage is essential for maintenance of genomic integrity. When DNA repair is compromised, mutations and genetic alterations that are the hallmarks of cancer cells can occur. One group of proteins important for repair of DNA damage is the RecQ family of DNA helicases. In humans, mutations in three of the five RecQ-like proteins (BLM, WRN, RTS) are associated with heritable cancer predisposition diseases and/or premature aging (Bloom, Werner, or Rothmund-Thomson syndromes, respectively). In the budding yeast Saccharomyces cerevisiae, the RecQ helicase, Sgs1, when mutated, exhibits many aspects of the phenotype associated with alterations in its human homologs such as genomic instability and premature aging (reviewed in Bernstein et al. 2010).Sgs1 and its homologs function in the homologous recombination (HR) DNA repair pathway. When a doublestrand break (DSB) occurs, the broken DNA ends are bound by the Mre11-Rad50-Xrs2 (MRX)/MRE11-RAD50-NBS1 (MRN) complex in yeast and humans, respectively (hereafter, the yeast nomenclature is written first and the human protein second). Subsequently, Sae2/CtIP, together with MRX/MRN, initiate resection of the DNA ends, thus preventing the binding of the Ku70-Ku80 heterodimer and inhibiting nonhomologous end joining (NHEJ) (Mimitou and Symington 2010;Foster et al. 2011;. Sae2 is essential to remove covalent adducts, such as Spo11, from DNA ends, but its function in resection initiation at endonuclease-induced DSBs can be bypassed by downstream nucleases. The absence of Mre11 leads to a greater delay in resection initiation at endonucleaseinduced DSBs than that observed for Sae2 due to the role of the MRX complex in recruiting Sgs1, Dna2, and Exo1 to DNA ends (Mimitou and Symington 2010;Shim et al. 2010). Subsequent long-range resection of the 39 overhangs can then occur by one of two pathways: the first utilizes the Sgs1/BLM helicase in conjunction with the endonuclease Dna2/DNA2 and the second u...

Sgs1 and Sae2 promote telomere replication by limiting accumulation of ssDNA

“…NATURE COMMUNICATIONS | DOI: 10.1038/ncomms6004 ARTICLE that sgs1D664D allele is proficient in the resolution of telomere recombination intermediates but defective in resection 57 , these results indicate that Sgs1 also contributes to resection of eroded telomeres. Noteworthy, although it has an opposite effect on the kinetics of senescence, the sgs1D664D allele impaired the formation of type II survivors as did the full deletion of the gene 33 ( Fig.…”

“…To address this possibility, we used the sgs1D664D separation of functions allele 55 . This allele has a defect in the processivity of resection at DSBs 56,57 . In agreement with previous report 33 , we found that full deletion of SGS1 accelerated the loss of viability of est2D cells, although the defect was less pronounced in our strain background (Fig.…”

DNA Replication Stress Phosphoproteome Profiles Reveal Novel Functional Phosphorylation Sites on Xrs2 in Saccharomyces cerevisiae

“…SAE2 is required for activating Mre11 endonuclease activity (Cannavo and Cejka 2014). Given the strong genetic interaction with sae2D, we hypothesized that Thr675 on Xrs2 may function in a parallel pathway of activating Mre11 endonuclease activity, which is essential for timely removal of the MRX complex from DSB ends, and the retention of MRX results in prolonged checkpoint activation and growth defects (Clerici et al 2006;Bernstein et al 2013;Chen et al 2015). Our genetic studies showed that tel1D and mec1D checkpoint mutants suppressed the severe sensitivity of an xrs2 S349A, T675A sae2D strain to MMS ( Figure 5B), consistent with the idea that prolonged checkpoint activation is responsible for the severe MMS-sensitivity phenotype.…”

“…The Mre11 complex plays a parallel role with Exonuclease 1 (Exo1) in producing long-range ssDNA tails (Bernstein et al 2013;Cannavo and Cejka 2014). The genetic interaction of xrs2 S349A, T675A with exo1D raises the possibility that The genetic interactions with tof1 S379A, S626A and xrs2 S349A, T675A were selected from the manually curated genetic interactions with tof1D and xrs2D in the Saccharomyces Genome Database (http://www.yeastgenome.org/).…”

“…The Mre11 endonuclease activity (promoted by Sae2) is known to be required for removing the MRX complex from DSB ends to facilitate subsequent long-range resection (Clerici et al 2006;Bernstein et al 2013;Chen et al 2015). Retention of MRX on the damage site results in prolonged checkpoint activation and growth defects (Clerici et al 2006;Chen et al 2015).…”