Kinetic Basis of Nucleotide Selection Employed by a Protein Template-Dependent DNA Polymerase
Kinetic Basis of Nucleotide Selection Employed by a Protein Template-Dependent DNA Polymerase
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DOI：
10.1021/bi100433x
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发表时间：
2010-07-06
期刊：
影响因子：
2.9
通讯作者：
Suo, Zucai
中科院分区：
文献类型：
--
作者：
Brown, Jessica A.;Fowler, Jason D.;Suo, Zucai
Rev1是一种y家族DNA聚合酶，参与自发和DNA损伤诱导的诱变事件。在本文中，我们采用预稳态动力学方法建立了人类Rev1选择核苷酸的动力学基础，Rev1是一种独特的核苷酸基转移酶，利用蛋白质模板导向机制优先指导dCTP的结合。这项工作表明，dCTP的高掺入效率取决于两个底物：一个传入的dCTP和一个模板基dG。人类Rev1极低的碱基替换保真度（100至10(-5)）是由于dCTP与模板碱基dA、dT和dC的误结合优于正确的dntp。使用非天然核苷酸类似物，我们发现人类Rev1残基R357与进入的dNTP之间的氢键相互作用对于DNA合成不是必需的。最后，人类Rev1主要通过降低掺入率来区分核糖核苷酸和脱氧核糖核苷酸，并且人类Rev1的糖选择性对核糖核苷酸的2'-取代基的大小和取向都很敏感。
Rev1, a Y-family DNA polymerase, contributes to spontaneous and DNA damage-induced mutagenic events. In this paper, we have employed pre-steady-state kinetic methodology to establish a kinetic basis for nucleotide selection by human Rev1, a unique nucleotidyl transferase that uses a protein template-directed mechanism to preferentially instruct dCTP incorporation. This work demonstrated that the high incorporation efficiency of dCTP is dependent on both substrates: an incoming dCTP and a templating base dG. The extremely low base substitution fidelity of human Rev1 (100 to 10(-5)) was due to the preferred misincorporation of dCTP with templating bases dA, dT, and dC over correct dNTPs. Using non-natural nucleotide analogues, we showed that hydrogen bonding interactions between residue R357 of human Rev1 and an incoming dNTP are not essential for DNA synthesis. Lastly, human Rev1 discriminates between ribonucleotides and deoxyribonucleotides mainly by reducing the rate of incorporation, and the sugar selectivity of human Rev1 is sensitive to both the size and orientation oldie 2'-substituent of a ribonucleotide.