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Journal of Nucleic Acids
Volume 2010, Article ID 101495, 10 pages
http://dx.doi.org/10.4061/2010/101495
Research Article

Replication Past the š›¾ -Radiation-Induced Guanine-Thymine Cross-Link G[8,5-Me]T by Human and Yeast DNA Polymerase šœ‚

Department of Chemistry, University of Connecticut, Storrs, CT 06269, USA

Received 9 June 2010; Accepted 2 July 2010

Academic Editor: Shigenori Iwai

Copyright © 2010 Paromita Raychaudhury and Ashis K. Basu. 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

š›¾ -Radiation-induced intrastrand guanine-thymine cross-link, G[8,5-Me]T, hinders replication in vitro and is mutagenic in mammalian cells. Herein we report in vitro translesion synthesis of G[8,5-Me]T by human and yeast DNA polymerase šœ‚ (hPol šœ‚ and yPol šœ‚ ). dAMP misincorporation opposite the cross-linked G by yPol šœ‚ was preferred over correct incorporation of dCMP, but further extension was 100-fold less efficient for G āˆ— :A compared to G āˆ— :C. For hPol šœ‚ , both incorporation and extension were more efficient with the correct nucleotides. To evaluate translesion synthesis in the presence of all four dNTPs, we have developed a plasmid-based DNA sequencing assay, which showed that yPol šœ‚ was more error-prone. Mutational frequencies of yPol šœ‚ and hPol šœ‚ were 36% and 14%, respectively. Targeted G ā†’ T was the dominant mutation by both DNA polymerases. But yPol šœ‚ induced targeted G ā†’ T in 23% frequency relative to 4% by hPol šœ‚ . For yPol šœ‚ , targeted G ā†’ T and G ā†’ C constituted 83% of the mutations. By contrast, with hPol šœ‚ , semi-targeted mutations (7.2%), that is, mutations at bases near the lesion, occurred at equal frequency as the targeted mutations (6.9%). The kind of mutations detected with hPol šœ‚ showed significant similarities with the mutational spectrum of G[8,5-Me]T in human embryonic kidney cells.