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Ku80 removal from DNA through double strand break–induced ubiquitylation

¹ Laboratory of Chromosome and Cell Biology and ² Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, NY 10065
³ Department of Molecular Biology, Weill Cornell Graduate School of Biomedical Sciences, Cornell Medical School, New York, NY 10021

Correspondence to Lisa Postow: PostowL{at}rockefeller.edu; or Hironori Funabiki: funabih{at}rockefeller.edu

The Ku70/Ku80 heterodimer, or Ku, is the central component of the nonhomologous end joining (NHEJ) pathway of double strand break (DSB) repair. Because Ku forms a ring through which the DSB threads, it likely becomes topologically attached to DNA during repair. The mechanism for its removal was unknown. Using a method to identify proteins recruited to DSBs in Xenopus laevis egg extract, we show that DSB-containing DNAs accumulate members of the Skp1–Cul1–F-box complex and K48-linked polyubiquitylated proteins in addition to known repair proteins. We demonstrate that Ku80 is degraded in response to DSBs in a ubiquitin-mediated manner. Strikingly, K48-linked polyubiquitylation, but not proteasomal degradation, is required for the efficient removal of Ku80 from DNA. This removal is DNA length dependent, as Ku80 is retained on duplex oligonucleotides. Finally, NHEJ completion and removal of Ku80 from DNA are independent from one another. We propose that DSB-induced ubiquitylation of Ku80 provides a mechanism to efficiently eliminate Ku from DNA for pre- and postrepair processes.

Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3-related; DB, double biotin; DNA-PKcs, DNA-dependent protein kinase catalytic subunit; DSB, double strand break; ms/ms, tandem mass spectrometry; NHEJ, nonhomologous end joining; SB, single biotin; SCF, Skp1–Cul1–F-box.

© 2008 Postow et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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