Nucleic Acids Research Advance Access originally published online on June 18, 2007
Nucleic Acids Research 2007 35(13):4275-4288; doi:10.1093/nar/gkm416
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Nucleic Acids Research, 2007, Vol. 35, No. 13 4275-4288
© 2007 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.
Structural Biology |
Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase
1Department of Biology and 2Department of Chemistry, New York University, New York, NY and 3Department of Biochemistry, Duke University Medical Center, Durham, NC, USA
*To whom correspondence should be addressed. Tel: (212)998-8231; Fax: (212)995-4015; Email: broyde{at}nyu.edu
Received January 10, 2007. Revised May 8, 2007. Accepted May 8, 2007.
We have investigated how a benzo[a]pyrene-derived N2-dG adduct, 10S(+)-trans-anti-[BP]-N2-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.
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