A DNA nick at Ku-blocked double-strand break ends serves as an entry site for exonuclease 1 (Exo1) or Sgs1–Dna2 in long-range DNA end resection

Weibin Wang, James M. Daley, Youngho Kwon, Xiaoyu Xue, Danielle S. Krasner, Adam S. Miller, Kevin A. Nguyen, Elizabeth A. Williamson, Eun Yong Shim, Sang Eun Lee, Robert Hromas, Patrick Sung

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

The repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is initiated by nucleolytic resection of the DNA break ends. The current model, being based primarily on genetic analyses in Saccharomyces cerevisiae and companion biochemical reconstitution studies, posits that end resection proceeds in two distinct stages. Specifically, the initiation of resection is mediated by the nuclease activity of the Mre11–Rad50 –Xrs2 (MRX) complex in conjunction with its cofactor Sae2, and long-range resection is carried out by exonuclease 1 (Exo1) or the Sgs1–Top3–Rmi1–Dna2 ensemble. Using fully reconstituted systems, we show here that DNA with ends occluded by the DNA end-joining factor Ku70 –Ku80 becomes a suitable substrate for long-range 5–3 resection when a nick is introduced at a locale proximal to one of the Ku-bound DNA ends. We also show that Sgs1 can unwind duplex DNA harboring a nick, in a manner dependent on a species-specific interaction with the ssDNA-binding factor replication protein A (RPA). These biochemical systems and results will be valuable for guiding future endeavors directed at delineating the mechanistic intricacy of DNA end resection in eukaryotes.

Original languageEnglish (US)
Pages (from-to)17061-17069
Number of pages9
JournalJournal of Biological Chemistry
Volume293
Issue number44
DOIs
StatePublished - Nov 2 2018

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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