title: Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection.
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.
authors.affiliation: From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and.
authors.forename: Danielle S
authors.initials: DS
authors.lastname: Krasner
authors.affiliation: From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and.
authors.forename: James M
authors.initials: JM
authors.lastname: Daley
authors.affiliation: From the Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520 and patrick.sung@yale.edu.
authors.forename: Patrick
authors.initials: P
authors.lastname: Sung
authors.affiliation: the Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana 47405 hniu@indiana.edu.
authors.forename: Hengyao
authors.initials: H
authors.lastname: Niu
mesh_terms: D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage / Q000235:genetics; D059766:DNA End-Joining Repair / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D059765:Homologous Recombination / Q000235:genetics*; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; C081647:high affinity DNA-binding factor, S cerevisiae; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I
keywords: DNA damage; DNA damage response; DNA-binding protein; Exo1; Ku; RPA; Saccharomyces cerevisiae; homologous recombination; non-homologous DNA end joining