TY - JOUR
T1 - Mechanism of the ATP-dependent DNA endresection machinery from Saccharomyces cerevisiae
AU - Niu, Hengyao
AU - Chung, Woo Hyun
AU - Zhu, Zhu
AU - Kwon, Youngho
AU - Zhao, Weixing
AU - Chi, Peter
AU - Prakash, Rohit
AU - Seong, Changhyun
AU - Liu, Dongqing
AU - Lu, Lucy
AU - Ira, Grzegorz
AU - Sung, Patrick
N1 - Funding Information:
Acknowledgements We thank J. Campbell, S. Brill and L. Symington for providing materials, X. Xue for the double Holliday junction substrate and S. Kowalczykowski for communicating results. This work was supported by grants from the US National Institutes of Health and by a postdoctoral fellowship from the Susan G. Komen for the Cure Foundation.
PY - 2010/9/2
Y1 - 2010/9/2
N2 - 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 phenotype1,2. DSB ends are resected in a 59 to 39 fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination2. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins3. Being guided by recent genetic studies4-6, 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 observations6, 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 recombination2,7, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role ofRPA, in the sequestration of ssDNAgenerated by DNA unwinding, enhancement of 59 strand incision, and protection of the 39 strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.
AB - 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 phenotype1,2. DSB ends are resected in a 59 to 39 fashion in cells, to yield single-stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination2. The resection process involves redundant pathways consisting of nucleases, DNA helicases and associated proteins3. Being guided by recent genetic studies4-6, 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 observations6, 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 recombination2,7, is not needed for resection either in cells or in the reconstituted system. Our results also unveil a multifaceted role ofRPA, in the sequestration of ssDNAgenerated by DNA unwinding, enhancement of 59 strand incision, and protection of the 39 strand. Our reconstituted system should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.
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U2 - 10.1038/nature09318
DO - 10.1038/nature09318
M3 - Article
C2 - 20811460
AN - SCOPUS:77956302112
SN - 0028-0836
VL - 467
SP - 108
EP - 111
JO - Nature
JF - Nature
IS - 7311
ER -