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Mechanism and Regulation of the Helicase‐driven Path of DNA End Resection in Saccharomyces cerevisiae
Author(s) -
Niu Hengyao,
Chen Xuefeng,
Kwon Youngho,
Chung Woo-Hyun,
Zhu Zhu,
Ira Grzegorz,
Sung Patrick
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.536.7
Subject(s) - helicase , saccharomyces cerevisiae , cyclin dependent kinase 1 , microbiology and biotechnology , homologous recombination , biology , dna , replication protein a , homology directed repair , nuclease , dna damage , dna repair , cell cycle , yeast , genetics , dna mismatch repair , dna binding protein , cell , gene , transcription factor , rna
The ends of DNA double‐strand breaks (DSBs) are resected in a cell‐cycle dependent fashion, to yield 3′ overhanging single‐stranded DNA (ssDNA) for the recruitment of factors critical for DNA damage checkpoint activation and repair by homologous recombination. As defined in studies in the budding yeast Saccharomyces cerevisiae , the DNA end resection process involves multiple, redundant pathways, one of which entails the Sgs1 helicase and its partners Rmi1‐Top3, Dna2 helicase/nuclease, Mre11‐Rad50‐Xrs2 (MRX) complex, and the heterotrimeric ssDNA‐binding protein RPA. We have reconstituted the DNA end resection reaction using the aforementioned factors. To gain further insight into how Cdk1 kinase activity regulates the motor‐driven resection pathway, we have purified the Cdk1‐Clb2 complex and examined its effects both in our reconstituted system and in conjunction with genetic analysis. Our studies indicate that one means of Cdk1 regulation is via direct phosphorylation of Dna2 at three consensus sites located within its amino‐terminus, which promotes efficient recruitment of Dna2 to DSBs. However, phospho‐mimic mutations of Dna2 cannot fully bypass the requirement of Cdk1 activity in vivo , raising the possibility that Cdk1 also targets other factor(s), an idea that is being examined using our reconstituted system. The biochemical and genetic systems that we have established should serve as a useful model for delineating the mechanistic intricacy of the DNA break resection process in eukaryotes.