Interplay between Ku and RPA in the restriction of Exo1-mediated DNA break end resection [DNA and Chromosomes]

June 11th, 2015 by Krasner, D. S., Daley, J. M., Sung, P., Niu, H.

DNA double-strand breaks can be eliminated via non-homologous end joining (NHEJ) or homologous recombination (HR). NHEJ is initiated by the association of Ku with DNA ends. In contrast, HR entails nucleolytic resection of the 5' strands, forming 3' ssDNA tails that become coated by 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 address these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protects against Exo1 in a manner that requires its DNA end binding activity. Despite binding poorly to ssDNA, Ku can nonetheless engage a 5' recessed DNA end with a 40-nt ssDNA overhang, where it localizes to the ss-dsDNA junction and efficiently blocks resection by Exo1. Interestingly, RPA can exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restore processing by Exo1. However, at a 40-nt tail, Ku remains stably associated at the ss-dsDNA junction, and RPA simultaneously engages 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.