Yasui Manabu, Suenaga Emi, Koyama Naoki, Masutani Chikahide, Hanaoka Fumio, Gruz Petr, Shibutani Shinya, Nohmi Takehiko, Hayashi Makoto, Honma Masamitsu
Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya, Tokyo 158-8501, Japan.
J Mol Biol. 2008 Apr 4;377(4):1015-23. doi: 10.1016/j.jmb.2008.01.033. Epub 2008 Jan 18.
Chronic inflammation involving constant generation of nitric oxide (*NO) 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 *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 kappaDeltaC (a truncated form of pol kappa) 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 kappaDeltaC, 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 kappaDeltaC 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 ()NO-induced adduct may play an important role in initiating inflammation-driven carcinogenesis.
巨噬细胞持续产生一氧化氮（NO）所导致的慢性炎症已被确认为与致癌作用相关的一个因素。在炎症部位，亚硝化脱氨基的DNA加合物，如2'-脱氧肌苷（dI）和2'-脱氧黄苷，主要由NO形成，并且可能与癌症的发生发展有关。在本研究中，我们使用一种新的荧光方法来分析跨损伤合成，从而探究Y家族DNA聚合酶（pols）产生的dI损伤的错配特性。在人DNA pols催化的引物延伸反应中，将含有单个dI损伤的寡脱氧核苷酸用作模板，以探究dI加合物的错配潜力。在dI加合物位点之前，由polα催化的引物延伸反应略有延迟；大多数引物延伸越过了该损伤。Polη和polκDeltaC（polκ的截短形式）很容易绕过dI损伤。通过使用两相聚丙烯酰胺凝胶电泳（PAGE）分析完全延伸的产物，以量化在损伤位点发生的错配频率和特异性。所有的聚合酶，即polα、polη和polκDeltaC，都促进了2'-脱氧胞苷单磷酸（dCMP），即错误的碱基，在dI损伤对面的优先掺入。令人惊讶的是，在损伤对面未观察到正确的碱基2'-脱氧胸苷单磷酸的掺入。对polα、polη和polκDeltaC进行的稳态动力学研究表明，dCMP优先在dI损伤对面掺入。这些聚合酶通过在损伤对面掺入dCMP绕过损伤，并延伸越过损伤。这些越过dC:dI碱基对的相对绕过频率比dT:dI碱基对的至少高3个数量级。因此，dI加合物是一种能够产生A→G转换的高度错配损伤。这种（*）NO诱导的加合物可能在引发炎症驱动的致癌作用中起重要作用。
