4022–4031 Nucleic Acids Research, 2008, Vol. 36, No. 12 doi:10.1093/nar/gkn344 Published online 31 May 2008 A mechanism for DNA-PK activation requiring unique contributions from each strand of a DNA terminus and implications for microhomology-mediated nonhomologous DNA end joining Katherine S. Pawelczak1 and John J. Turchi1,2,* 1Department of Biochemistry and Molecular Biology and 2Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46220, USA Received August 2, 2007; Revised May 6, 2008; Accepted May 12, 2008 ABSTRACT DNA-dependent protein kinase (DNA-PK) is an essential component of the nonhomologous end joining pathway (NHEJ), responsible for the repair of DNA double-strand breaks. Ku binds a DSB and recruits the catalytic subunit, DNA-PKcs, where it is activated once the kinase is bound to the DSB. The precise mechanism by which DNA activates DNA-PK remains unknown. We have investigated the effect of DNA structure on DNA- PK activation and results demonstrate that in Ku- dependent DNA-PKcs reactions, DNA-PK activation with DNA effectors containing two unannealed ends was identical to activation observed with fully duplex DNA effectors of the same length. The presence of a 6-base single-stranded extension resulted in decreased activation compared to the fully duplex DNA. DNA-PK activation using DNA effectors with compatible termini displayed increased activity compared to effectors with non- compatible termini. A strand orientation preference was observed in these reactions and suggests a model where the 3’ strand of the terminus is responsible for annealing and the 5’ strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in micro- homology-mediated NHEJ. INTRODUCTION DNA double-strand breaks (DSB) can be induced by ionizing radiation (IR), certain chemical compounds and collapsed replication forks. If not repaired, these breaks can lead to genomic instability, chromosomal abnormal- ities and cell death. Nonhomologous end joining (NHEJ) is the pathway largely responsible for the repair of DSBs induced by IR (1,2). The DNA-PK holoenzyme comprised of the Ku DNA-binding domain and the catalytic subunit is the main complex initiating the NHEJ process. Ku ﬁrst binds to the DNA termini, whereby the ring-like structure of Ku encircles the DNA end (3). This structure can also account for the ability of Ku to translocate along the duplex DNA (4–6). DNA-PKcs then binds to the DNA terminus in a Ku-dependent manner. Formation of this complex on DNA results in activation of the serine/ threonine protein kinase enzymatic activity. DNA-PK has been demonstrated to phosphorylate a number of proteins in the NHEJ pathway (7–13). Importantly, only phosphorylation of Artemis and autophosphorylation of DNA-PKcs have been demonstrated to alter biological activity and NHEJ catalyzed repair (14,15). Structural analysis of DNA-PK has revealed an open region within the kinase that upon interaction with DNA induces a conformational change in the protein that may play a role in activation of the kinase (16). Further work modeled a passage way through the DNA–PK complex within the catalytic subunit that can accommodate DNA, and upon DNA threading through this channel, may serve to protect the free termini. Following DNA binding, the formation of a DNA-PK dimer has been proposed to create a synaptic region responsible for bringing the two *To whom correspondence should be addressed. Tel: +317 278 1996; Fax: +317 274 0396; Email: jturchi@iupui.edu 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.