4168–4177 Nucleic Acids Research, 2012, Vol. 40, No. 9 doi:10.1093/nar/gkr1231 Published online 5 January 2012 Visualization of a DNA-PK/PARP1 complex Laura Spagnolo1,2,*, Jody Barbeau 3 , Nicola J. Curtin3, Edward P. Morris4 and Laurence H. Pearl1,5 1Cancer Research UK DNA Repair Enzymes Group, The Institute of Cancer Research, London SW3 6JB, 2Institute of Structural Molecular Biology, University of Edinburgh, EH9 3JR, 3Northern Institute for Cancer Research, Medical School, Newcastle University, Newcastle upon Tyne, 4Structural Electron Microscopy Group, Section of Structural Biology, The Institute of Cancer Research, London SW3 6JB and 5School of Life Sciences, University of Sussex, Brighton, BN1 9RH, UK Received June 16, 2011; Revised November 23, 2011; Accepted November 24, 2011 ABSTRACT The DNA-dependent protein kinase (DNA-PK) and Poly(ADP-ribose) polymerase-1 (PARP1) are critical enzymes that reduce genomic damage caused by DNA lesions. They are both activated by DNA strand breaks generated by physiological and envir- onmental factors, and they have been shown to interact. Here, we report in vivo evidence that DNA-PK and PARP1 are equally necessary for rapid repair. We purified a DNA-PK/PARP1 complex loaded on DNA and performed electron microscopy and single particle analysis on its tetra- meric and dimer-of-tetramers forms. By comparison with the DNA-PK holoenzyme and fitting crystal- lographic structures, we see that the PARP1 density is in close contact with the Ku subunit. Crucially, PARP1 binding elicits substantial con- formational changes in the DNA-PK synaptic dimer assembly. Taken together, our data support a func- tional, in-pathway role for DNA-PK and PARP1 in double-strand break (DSB) repair. We also propose a NHEJ model where protein–protein interactions alter substantially the architecture of DNA-PK dimers at DSBs, to trigger subsequent interactions or enzymatic reactions. INTRODUCTION DNA double-strand breaks (DSBs) are the most cytotoxic form of DNA damage. If unrepaired or incorrectly repaired, they can lead to apoptosis or genome instability. Two major DSB repair pathways exist: homologous recombination (HR) and non-homologous end joining (NHEJ) (1). In the presence of sister chromatids acting as templates for repair, DSBs can be repaired by HR. In NHEJ, the DNA broken ends are resected and/or pro- cessed and the DNA backbones ligated to restore strand continuity without the need for a template. The DNA-dependent protein kinase (DNA-PK) heterotrimeric enzyme is the early player in mammalian NHEJ (2). DNA-PK is formed by a DSB recognition module, called Ku (preassembled as a heterodimer of the Ku70 and Ku80 proteins) (3) and a large catalytic subunit (DNA-PKcs, 0.5 MDa) (4,5). After recognizing and binding to a DSB, Ku recruits the catalytic subunit via the C-terminal domain of the Ku80 subunit. DNA-PKcs is a serine/threonine kinase belonging to the phosphatidylinositol-3-OH kinase (PI3K)-related (PIKK) family. DNA-PK assembles on DNA as a bridging complex, where two heterotrimers maintain the two DNA broken ends in close proximity, providing a scaf- folding platform to recruit further NHEJ enzymes (6). These factors include Artemis and PNK, which are required to process the broken ends; X family polymer- ases, which promote microhomology and cohesion between the broken ends; DNA ligase IV-XRCC4, which closes the phospho-diester backbone on both strands, and the XLF/Cernunnos factor (7). Autophos- phorylation sites, crucial to NHEJ regulation, have been identiﬁed in DNA-PKcs at two main clusters, as well as within its catalytic domain (7). A recent electron micros- copy study visualized a substantial remodeling of DNA-PK on autophosphorylation (8). PARP enzymes use nicotinamide as a substrate to polymerize ADP-ribose moieties onto target proteins, a process called poly-ADP-ribosylation (or PARylation) (9). The best-studied PARP enzyme is PARP1, which has a key role in DNA repair and in particular single- strand break repair/base excision repair (10). PARP1 detects and binds single strand DNA breaks and then poly-ADP-ribosylates itself (auto-PARylation) and other *To whom correspondence should be addressed. Tel. +44 0 131 650 7066; Fax +44 0 131 650 8650; Email: laura.spagnolo@ed.ac.uk The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.