Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA adduct, during translesion synthesis catalyzed by human DNA polymerases
Miscoding properties of 2'-deoxyinosine, a nitric oxide-derived DNA adduct, during translesion synthesis catalyzed by human DNA polymerases
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DOI：
10.1016/j.jmb.2008.01.033
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发表时间：
2008-04-04
影响因子：
5.6
通讯作者：
Honma, Masamitsu
中科院分区：
文献类型：
--
作者：
Yasui, Manabu;Suenaga, Emi;Honma, Masamitsu
巨噬细胞持续产生一氧化氮((NO)-N中心点)的慢性炎症已被认为是与肿瘤发生有关的因素。在炎症部位，亚硝化脱氨基DNA加合物如2‘-脱氧肌苷(Di)和2’-脱氧黄嘌呤主要由中心点NO形成，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，探索了Y家族DNA聚合酶(POLS)产生的Di损伤的错误编码特性。以含有单个Di残基的寡核苷酸为模板，在人DNA Pols催化的引物延伸反应中探索Di加合物的错误编码可能性。Polα催化的引物延伸反应在Di加合物位点之前略有延迟；大多数引物延伸到病变之外。POLETA和POLKAPPA Delta C(POLK的截短形式)很容易绕过Di损伤。使用两阶段PAGE对完全延伸的产物进行分析，以量化病变部位发生错误编码的频率和特异性。所有的Pols，即polAlpha、polETA和polkappa Delta C，都促进2‘-脱氧胞苷单磷酸(DCMP)优先掺入与Di损伤相反的错误碱基。令人惊讶的是，在病变对面没有观察到正确的碱基2-脱氧胸苷单磷酸的掺入。用polAlpha、polETA和Pol kappa Delta C进行的稳态动力学研究表明，dCMP优先结合在Di损伤的对面。这些POL通过加入与病变相对的dCMP而绕过病变，并延伸到病变之外。这些通过DC：DI对的相对旁路频率比DT：DI对的至少高3个数量级。因此，Di加合物是一种高度错误编码的损伤，能够产生A-&G转换。这种中心点NO诱导的加合物可能在启动炎症驱动的致癌过程中发挥重要作用。(C)2008爱思唯尔有限公司。保留所有权利。
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.