title: Mechanism of the ATP-dependent DNA end-resection machinery from Saccharomyces cerevisiae.
abstract: If not properly processed and repaired, DNA double-strand breaks (DSBs) can give rise to deleterious chromosome rearrangements, which could ultimately lead to the tumour phenotype. DSB ends are resected in a 5' to 3' fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins. Being guided by recent genetic studies, we have reconstituted the first eukaryotic ATP-dependent DNA end-resection machinery comprising the Saccharomyces cerevisiae Mre11-Rad50-Xrs2 (MRX) complex, the Sgs1-Top3-Rmi1 complex, Dna2 protein and the heterotrimeric ssDNA-binding protein RPA. Here we show that DNA strand separation during end resection is mediated by the Sgs1 helicase function, in a manner that is enhanced by Top3-Rmi1 and MRX. In congruence with genetic observations, although the Dna2 nuclease activity is critical for resection, the Mre11 nuclease activity is dispensable. By examining the top3 Y356F allele and its encoded protein, we provide evidence that the topoisomerase activity of Top3, although critical for the suppression of crossover recombination, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role of RPA, in the sequestration of ssDNA generated by DNA unwinding, enhancement of 5' strand incision, and protection of the 3' strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
authors.forename: Hengyao
authors.initials: H
authors.lastname: Niu
authors.forename: Woo-Hyun
authors.initials: WH
authors.lastname: Chung
authors.forename: Zhu
authors.initials: Z
authors.lastname: Zhu
authors.forename: Youngho
authors.initials: Y
authors.lastname: Kwon
authors.forename: Weixing
authors.initials: W
authors.lastname: Zhao
authors.forename: Peter
authors.initials: P
authors.lastname: Chi
authors.forename: Rohit
authors.initials: R
authors.lastname: Prakash
authors.forename: Changhyun
authors.initials: C
authors.lastname: Seong
authors.forename: Dongqing
authors.initials: D
authors.lastname: Liu
authors.forename: Lucy
authors.initials: L
authors.lastname: Lu
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Ira
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000378:metabolism; D004260:DNA Repair; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D053484:RecQ Helicases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A; C502206:Rmi1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C502374:TOP3 protein, S cerevisiae; D000255:Adenosine Triphosphate; C090472:SGS1 protein, S cerevisiae; D004265:DNA Helicases; C410222:DNA2 protein, S cerevisiae; D053484:RecQ Helicases
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