breakrepair.PDFVIP

Organization and dynamics of the nonhomologous PNAS PLUS end-joining machinery during DNA double-strand break repair Dylan A. Reida, Sarah Keegana, Alejandra Leo-Maciasa, Go Watanabeb, Natasha T. Strandec, Howard H. Changb, Betul Akgol Oksuza, David Fenyoa, Michael R. Lieberb, Dale A. Ramsdenc, and Eli Rothenberga,1 aDepartment of Biochemistry and Molecular Pharmacology, New York University School of Medicine, New York, NY 10016; bDepartment of Pathology, Biochemistry Molecular Biology, Molecular Microbiology Immunology, USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA 90089; and cDepartment of Biochemistry and Biophysics, Lineberger Comprehensive Cancer Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599 Edited by Tanya T. Paull, University of Texas, Austin, TX, and accepted by the Editorial Board April 9, 2015 (received for review October 20, 2014) Nonhomologous end-joining (NHEJ) is a major repair pathway for structures in vitro (26–28). Whether such filaments mediate re- DNA double-strand breaks (DSBs), involving synapsis and ligation pair in vivo has not yet been determined. of the broken strands. We describe the use of in vivo and in vitro Our present understanding of the cellular NHEJ response to single-molecule methods to define the organization and interac- DSBs is based primarily on in vitro biochemical and structural tion of NHEJ repair proteins at DSB ends. Super-resolution fluores- studies done with purified proteins, together with cellular ob- cence microscopy allowed the precise visualization of XRCC4, XLF, servations in which a radiologic or pharmacologic stimulus dam- and DNA ligase IV filaments adjacent to DSBs, which bridge the ages DNA, allowing observation of the repair process (29–31). broken chromosome and direct rejoining. We show, by single- Typically, cellular as