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Journal of Nucleic Acids
Volume 2010 (2010), Article ID 823917, 11 pages
http://dx.doi.org/10.4061/2010/823917
Research Article

Coincident In Vitro Analysis of DNA-PK-Dependent and -Independent Nonhomologous End Joining

1Radiology & Imaging Sciences Department, Nuclear Medicine Section, Warren G. Magnuson Clinical Center, National Institutes of Health, Bethesda, MD 20892, USA
2Applied Biosystems, Advanced Genetic Applications, 500 Cummiing Center, Suite 2450, Beverly, MA 01915, USA
3Molecular Genetics Department, Medical University of Lodz, Lodz 92-215, Poland

Received 15 May 2010; Accepted 6 June 2010

Academic Editor: Ashis Basu

Copyright © 2010 Cynthia L. Hendrickson et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In mammalian cells, DNA double-strand breaks (DSBs) are primarily repaired by nonhomologous end joining (NHEJ). The current model suggests that the Ku 70/80 heterodimer binds to DSB ends and recruits DNA- to form the active DNA-dependent protein kinase, DNA-PK. Subsequently, XRCC4, DNA ligase IV, XLF and most likely, other unidentified components participate in the final DSB ligation step. Therefore, DNA-PK plays a key role in NHEJ due to its structural and regulatory functions that mediate DSB end joining. However, recent studies show that additional DNA-PK-independent NHEJ pathways also exist. Unfortunately, the presence of DNA- appears to inhibit DNA-PK-independent NHEJ, and in vitro analysis of DNA-PK-independent NHEJ in the presence of the DNA- protein remains problematic. We have developed an in vitro assay that is preferentially active for DNA-PK-independent DSB repair based solely on its reaction conditions, facilitating coincident differential biochemical analysis of the two pathways. The results indicate the biochemically distinct nature of the end-joining mechanisms represented by the DNA-PK-dependent and -independent NHEJ assays as well as functional differences between the two pathways.