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J Biol Chem. 2015 Jul 24;290(30):18806-16. doi: 10.1074/jbc.M115.660191. Epub 2015 Jun 11.

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection.

Author information

1
From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and.
2
From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and patrick.sung@yale.edu.
3
the Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana 47405 hniu@indiana.edu.

Abstract

DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5'-strands, forming 3'-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5'-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process.

KEYWORDS:

DNA damage; DNA damage response; DNA-binding protein; Exo1; Ku; RPA; Saccharomyces cerevisiae; homologous recombination; non-homologous DNA end joining

PMID:
26067273
PMCID:
PMC4513135
DOI:
10.1074/jbc.M115.660191
[Indexed for MEDLINE]
Free PMC Article

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