期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 377, 期 4, 页码 1015-1023出版社
ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2008.01.033
关键词
inflammation; nitric oxide; DNA adduct; translesion synthesis; nonradioactive analysis
Chronic inflammation involving constant generation of nitric oxide ((NO)-N-center dot) by macrophages has been recognized as a factor related to carcinogenesis. At the site of inflammation, nitrosatively deaminated DNA adducts such as 2'-deoxyinosine (dI) and 2'-deoxyxanthosine are primarily formed by center dot NO and may be associated with the development of cancer. In this study, we explored the miscoding properties of the dI lesion generated by Y-family DNA polymerases (pols) using a new fluorescent method for analyzing translesion synthesis. An oligodeoxynucleotide containing a single dI lesion was used as a template in primer extension reaction catalyzed by human DNA pols to explore the miscoding potential of the dI adduct. Primer extension reaction catalyzed by pol alpha was slightly retarded prior to the dI adduct site; most of the primers were extended past the lesion. Pol eta and pol kappa Delta C (a truncated form of pol K) readily bypassed the dI lesion. The fully extended products were analyzed by using two-phased PAGE to quantify the miscoding frequency and specificity occurring at the lesion site. All pols, that is, pol alpha, pol eta, and pol kappa Delta C, promoted preferential incorporation of 2'-deoxycytidine monophosphate (dCMP), the wrong base, opposite the dI lesion. Surprisingly, no incorporation of 2-deoxythymidine monophosphate, the correct base, was observed opposite the lesion. Steady-state kinetic studies with pol alpha, pol eta, and Pol kappa Delta C indicated that dCMP was preferentially incorporated opposite the dI lesion. These pols bypassed the lesion by incorporating dCMP opposite the lesion and extended past the lesion. These relative bypass frequencies past the dC:dI pair were at least 3 orders of magnitude higher than those for the dT:dI pair. Thus, the dI adduct is a highly miscoding lesion capable of generating A -> G transition. This center dot NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis. (C) 2008 Elsevier Ltd. All rights reserved.
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