Effects of a guanine-derived formamidopyrimidine lesion on DNA replication - Translesion DNA synthesis, nucleotide insertion, and extension kinetics
Effects of a guanine-derived formamidopyrimidine lesion on DNA replication - Translesion DNA synthesis, nucleotide insertion, and extension kinetics
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DOI：
10.1074/jbc.m200316200
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发表时间：
2002-04-26
影响因子：
4.8
通讯作者：
Ide, H
中科院分区：
文献类型：
--
作者：
Asagoshi, K;Terato, H;Ide, H
鸟嘌呤衍生的2，6-二氨基-4-羟基-5-甲酰胺基嘧啶（FapyG）是由活性氧形成的主要DNA损伤。在这项研究中，一个定义的寡核苷酸模板含有5-N-甲基化类似物的FapyG（mFapyG）的制备，并在体外定量评估其对DNA复制的影响。结果进一步与7，8-二氢-8-氧代鸟嘌呤和无嘌呤/无嘧啶位点嵌入在相同的序列背景下获得的结果进行了比较。mFapyG对由大肠杆菌DNA聚合酶I Klenow片段催化的DNA合成构成相当强的但不是绝对的阻断，其具有或不具有相关的3 '-5'外切核酸酶活性，从而允许以有限的效率进行跨损伤合成。跨损伤合成效率为G > 7，8-二氢-8-氧代鸟嘌呤> mFapyG >脱嘌呤/脱嘧啶位点。对mFapyG的核苷酸插入（f（ins）= V-max/K-m用于插入）和延伸（f（ext）= V-max/K-m用于延伸）效率的分析表明，延伸步骤构成DNA合成的主要动力学屏障。当绕过mFapyG时，dCMP（一种同源核苷酸）优先插入病变的对面（dCMP（相对f（ins）= 1）>> dTMP（2.4 x 10（-4））近似于dAMP（8.1 x 10（-5））> dGMP（4.5 × 10（-7），并且含有mFapyG：C对的引物末端最有效地延伸（mFapyG：C（相对f（ext）= 1）> mFapyG：T（4.6 × 10（-3））>> mFapyG：A和mFapyG：G（未观察到延伸））。因此，mFapyG是一种潜在的致死性病变，但不是致突变前病变。
2,6-Diamino-4-hydroxy-5-formamidopyrimidine derived from guanine (FapyG) is a major DNA lesion formed by reactive oxygen species. In this study, a defined oligonucleotide template containing a 5-N-methylated analog of FapyG (mFapyG) was prepared, and its effect on DNA replication was quantitatively assessed in vitro. The results were further compared with those obtained for 7,8-dihydro-8-oxoguanine and an apurinic/apyrimidinic site embedded in the same sequence context. mFapyG constituted a fairly strong but not absolute block to DNA synthesis catalyzed by Escherichia coli DNA polymerase I Klenow fragment with and without an associated 3'-5' exonuclease activity, thereby permitting translesion synthesis with a limited efficiency. The efficiency of translesion synthesis was G > 7,8-dihydro-8-oxoguanine > mFapyG > apurinic/apyrimidinic site. Analysis of the nucleotide insertion (f(ins) = V-max/K-m for insertion) and extension (f(ext) = V-max/K-m for extension) efficiencies for mFapyG revealed that the extension step constituted a major kinetic barrier to DNA synthesis. When mFapyG was bypassed, dCMP, a cognate nucleotide, was preferentially inserted opposite the lesion (dCMP (relative f(ins) = 1) >> dTMP (2.4 x 10(-4)) approximate to dAMP (8.1 x 10(-5)) > dGMP (4.5 x 10(-7))), and the primer terminus containing a mFapyG:C pair was most efficiently extended (mFapyG:C (relative f(ext) = 1) > mFapyG:T (4.6 x 10(-3)) >> mFapyG:A and mFapyG:G (extension not observed)). Thus, mFapyG is a potentially lethal but not premutagenic lesion.