Nucleic Acids Research Advance Access originally published online on April 1, 2008
Nucleic Acids Research 2008 36(9):2895-2905; doi:10.1093/nar/gkn126
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Nucleic Acids Research, 2008, Vol. 36, No. 9 2895-2905
© 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.
Nucleic Acid Enzymes |
Tolerance for 8-oxoguanine but not thymine glycol in alignment-based gap filling of partially complementary double-strand break ends by DNA polymerase 
in human nuclear extracts
1Department of Pharmacology and Toxicology and Massey Cancer Center, Virginia Commonwealth University, Richmond, VA 23298, 2Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, 28049 Madrid, Spain and 3Laboratory of Molecular Genetics and Laboratory of Structural Biology, National Institute of Environmental Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA
*To whom correspondence should be addressed. Tel: +1 804 828 9640; Fax: +1 804 828 8079; Email: lpovirk{at}vcu.edu
Received December 11, 2007. Revised March 2, 2008. Accepted March 5, 2008.
Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3' overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase 
. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or -GTA) 3' overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote error-prone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase was replaced with that of polymerase β, could not substitute for polymerase in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.