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Proc Natl Acad Sci U S A. 2019 Mar 26;116(13):6091-6100. doi: 10.1073/pnas.1819276116. Epub 2019 Mar 8.

Regulatory control of Sgs1 and Dna2 during eukaryotic DNA end resection.

Author information

1
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032.
2
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, CT 06520.
3
Department of Biochemistry and Structural Biology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229.
4
Department of Pathology and Cell Biology, Columbia University, New York, NY 10032.
5
Department of Biochemistry & Molecular Biophysics, Columbia University, New York, NY 10032; ecg2108@cumc.columbia.edu.

Abstract

In the repair of DNA double-strand breaks by homologous recombination, the DNA break ends must first be processed into 3' single-strand DNA overhangs. In budding yeast, end processing requires the helicase Sgs1 (BLM in humans), the nuclease/helicase Dna2, Top3-Rmi1, and replication protein A (RPA). Here, we use single-molecule imaging to visualize Sgs1-dependent end processing in real-time. We show that Sgs1 is recruited to DNA ends through Top3-Rmi1-dependent or -independent means, and in both cases Sgs1 is maintained in an immoble state at the DNA ends. Importantly, the addition of Dna2 triggers processive Sgs1 translocation, but DNA resection only occurs when RPA is also present. We also demonstrate that the Sgs1-Dna2-Top3-Rmi1-RPA ensemble can efficiently disrupt nucleosomes, and that Sgs1 itself possesses nucleosome remodeling activity. Together, these results shed light on the regulatory interplay among conserved protein factors that mediate the nucleolytic processing of DNA ends in preparation for homologous recombination-mediated chromosome damage repair.

KEYWORDS:

DNA end resection; DNA repair; helicase; homologous recombination; single molecule

PMID:
30850524
PMCID:
PMC6442620
[Available on 2019-09-26]
DOI:
10.1073/pnas.1819276116

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