Fidelity of nucleotide insertion at 8-Oxo-7,8-dihydroguanine by mammalian DNA polymerase δ -: Steady-state and pre-steady-state kinetic analysis
Fidelity of nucleotide insertion at 8-Oxo-7,8-dihydroguanine by mammalian DNA polymerase δ -: Steady-state and pre-steady-state kinetic analysis
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DOI：
10.1074/jbc.m006696200
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发表时间：
2001-02-09
影响因子：
4.8
通讯作者：
Guengerich, FP
中科院分区：
文献类型：
--
作者：
Einolf, HJ;Guengerich, FP
通过稳态和稳态前快速猝灭动力学分析，检查了胎牛胸腺 DNA 聚合酶 δ (pol δ) 对 8-oxo-7,8-二氢鸟嘌呤 (8-oxoG) 的核苷酸插入。在与辅助蛋白增殖细胞核抗原 (PCNA) 的稳态反应中，pol δ 优先掺入与 8-oxoG 相反的 dCTP，其掺入效率比掺入未修饰 DNA 低一个数量级（主要是由于 K-m 增加）。与 8-oxoG 相对的掺入的前稳态动力学分析显示，dCTP 或 dATP 掺入的双相动力学，其速率与与 G 相对的 dCTP 掺入相似，硫代磷酸酯效应较大 (>100)，并且寡核苷酸解离在稳态下显然是限速的。尽管 pol δ 更倾向于掺入 dCTP（14% 的 dATP 错误掺入），但超过 A:8-oxoG 错配的延伸占主导地位。研究发现，与未修饰的 DNA 相比，PCNA 的存在对于 8-oxoG 加合物的核苷酸掺入来说是更重要的因素，使核苷酸掺入的稳态前速率增加了 > 2 个数量级，并且对于超出 8-oxoG 的核苷酸延伸至关重要。 8-oxoG 的 pol δ 复制保真度取决于 K-m,K-d(dNTP) 的贡献以及磷酸二酯键形成的速率，而 PCNA 是 8-oxoG 加合物掺入和延伸的重要辅助蛋白。
Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8- oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K-m). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased presteady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K-m,K-d(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.