Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells Farjana Fattah¤a, Eu Han Lee, Natalie Weisensel¤b, Yongbao Wang, Natalie Lichter, Eric A. Hendrickson* Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis, Minnesota, United States of America Abstract The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer- dependent or ‘‘classic’’ NHEJ (C-NHEJ) pathway and an ‘‘alternative’’ NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86- cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice. Citation: Fattah F, Lee EH, Weisensel N, Wang Y, Lichter N, et al. (2010) Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells. PLoS Genet 6(2): e1000855. doi:10.1371/journal.pgen.1000855 Editor: Christopher E. Pearson, The Hospital for Sick Children and University of Toronto, Canada Received July 31, 2009; Accepted January 23, 2010; Published February 26, 2010 Copyright: 2010 Fattah et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the NIH awards GM069576 and HL079559 to EAH. We would like to acknowledge the assistance of the Flow Cytometry Core Facility of the Masonic Cancer Center, a comprehensive cancer center designated by the National Cancer Institute, supported in part by P30 CA77598. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: hendr064@umn.edu ¤a Current address: Department of Pharmacology, Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas, United States of America ¤b Current address: School of Medicine and Public Health, University of Wisconsin, Madison, Wisconsin, United States of America Introduction One of the most harmful lesions a cell can encounter is a DNA double-strand break (DSB). In all organisms, efficient repair of these DSBs is critical for the maintenance of genomic integrity and viability [1]. Unfortunately, DSBs are frequently generated endogenously during normal cellular processes such as DNA replication, lymphoid V(D)J or class-switch recombination and are induced exogenously by the exposure to a variety of genotoxic agents such as ionizing radiation or chemotherapeutics [2]. Cells have conspired to meet this demand on their genetic material with the evolution of two mechanistically distinct pathways to repair DSBs: homologous recombination (HR), which takes advantage of either a homologous chromosome or a sister chromatid to join the broken DNA ends [3] and non-homologous end joining (NHEJ), a process that directly joins the DSB with little or no sequence homology between the broken ends [2]. In bacteria and lower eukaryotes, HR dominates the DNA DSB repair events whereas in higher eukaryotes, and especially in mammals, NHEJ is the preferred pathway for DNA DSB repair. NHEJ consists of at least two genetically and biochemically distinct sub-pathways: a main— ‘‘classic’’—end-joining pathway (C-NHEJ) and one interchange- ably referred to as microhomology-mediated end joining (MMEJ) [4], alternative NHEJ (A-NHEJ), or backup NHEJ (B-NHEJ) [5,6] (hereafter referred to as A-NHEJ). C-NHEJ, while by no means precise, results in minimal DNA end processing, whereas A-NHEJ mechanistically results in deletions per force that are often accompanied by microhomology at the repair junction {[7,8]; reviewed by [6,9]}. There are at least seven proteins required for C-NHEJ: Ku70, Ku86, the DNA dependent protein kinase catalytic subunit (DNA- PKcs), Artemis, X-ray cross complementing 4 (XRCC4), XRCC4- like factor (XLF) and DNA ligase IV (LIGIV) {reviewed by [10]}. The basic mechanism of C-NHEJ has been worked out in great detail. Ku70 and Ku86 form a heterodimer (Ku) that contains an internal cavity, which Ku uses to bind to and encircle broken DNA ends [11]. Ku, besides protecting DNA ends from exonucleolytic attack, also recruits DNA-PKcs, a phosphoinositol-3-like family serine/threonine protein kinase [12]. Together, Ku70, Ku86 and DNA-PKcs form the DNA-dependent protein kinase complex (DNA-PK) and the assembly of this trimeric complex on the ends of double-stranded DNA activates the kinase activity of DNA- PLoS Genetics | www.plosgenetics.org 1 February 2010 | Volume 6 | Issue 2 | e1000855