Kinetics of Deoxy-CTP Incorporation Opposite a dG-C8-N-2-Aminofluorene Adduct by a High-Fidelity DNA Polymerase
Kinetics of Deoxy-CTP Incorporation Opposite a dG-C8-N-2-Aminofluorene Adduct by a High-Fidelity DNA Polymerase
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DOI：
10.1016/j.jmb.2008.12.067
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发表时间：
2009-03-06
影响因子：
5.6
通讯作者：
Wagner, Jerome E.
中科院分区：
文献类型：
--
作者：
Burnouf, Dominique Y.;Wagner, Jerome E.
模型致癌物N-2-乙酰氨基芴与鸟嘌呤的C8位共价结合，形成两种加合物，N-（2 '-脱氧鸟苷-8-基）氨基芴（G-AF）和N-2-（2'-脱氧鸟苷-8-基）-乙酰氨基芴（G-AAF）。虽然它们在化学上密切相关，但它们的生物学效应截然不同，并且它们通过不同的损伤耐受途径进行加工。G-AF被复制型和高保真聚合酶绕过，我们使用嗜热芽孢杆菌菌株的DNA聚合酶I片段作为高保真聚合酶的模型来研究与单一G-AF相反的脱氧CTP（dCTP）掺入的动力学。改性三元复合物鉴定了这些三元复合物的两个群体：（i）较小的生产性部分（20%），其容易以与针对无加合物的三元复合物测量的速率类似的速率与G-AF加合物相反地掺入dCTP，和（ii）缓慢演变成生产性复合物的非生产性复合物的主要部分（80%）。根据结构数据，我们认为这种缓慢的速率反映了活性位点内修饰碱基从预插入位点到插入位点的易位。通过限制这种易位率，G-AF损伤揭示了在dNTP结合之后和化学之前发生的新的动力学步骤。(C)2009年由Elsevier Ltd.出版
The model carcinogen N-2-acetylaminofluorene covalently binds to the C8 position of guanine to form two adducts, the N-(2'-deoxyguanosine-8-yl)aminofluorene (G-AF) and the N-2-(2'-deoxyguanosine-8-yl)-acetylaminofluorene (G-AAF). Although they are chemically closely related, their biological effects are strongly different and they are processed by different damage tolerance pathways. G-AF is bypassed by replicative and high-fidelity polymerases, while specialized polymerases ensure synthesis past of G-AAF We used the DNA polymerase I fragment of a Bacillus slearothermophilus strain as a model for a high-fidetity polymerase to study the kinetics of incorporation of deoxy-CTP (dCTP) opposite a single G-AF Pre-steady-state kinetic experiments revealed a drastic reduction in dCTP incorporation performed by the G-AF-modified ternary complex. Two populations of these ternary complexes were identified: (i) a minor productive fraction (20%) that readily incorporates dCTP opposite the G-AF adduct with a rate similar to that measured for the adduct-free ternary complexes and (ii) a major fraction of unproductive complexes (80%) that slowly evolve into productive ones. lit the fight of structural data, we suggest that this slow rate reflects the translocation of the modified base within the active site, from the pre-insertion site into the insertion site. By making this translocation rate limiting, the G-AF lesion reveals a novel kinetic step occurring after dNTP binding and before chemistry. (C) 2009 Published by Elsevier Ltd.