Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase ζ
Substitution of a residue contacting the triphosphate moiety of the incoming nucleotide increases the fidelity of yeast DNA polymerase ζ
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DOI：
10.1093/nar/gkn023
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发表时间：
2008-03-01
影响因子：
14.9
通讯作者：
Washington, M. Todd
中科院分区：
文献类型：
--
作者：
Howell, Craig A.;Kondratick, Christine M.;Washington, M. Todd
DNA 聚合酶 zeta (pol zeta) 是 DNA 损伤诱导突变所需的酶，在多种 DNA 损伤的易错复制中发挥作用。在此过程中，pol zeta 从通过其他聚合酶掺入相反模板损伤的核苷酸延伸。与经典聚合酶不同，pol zeta 从包含错配或损伤的引物末端碱基对有效延伸，并且合成具有中等保真度的 DNA。在这里，我们描述了三种酵母 pol zeta 突变蛋白的遗传和生化研究，这些突变蛋白含有与引入核苷酸的三磷酸部分接触的高度保守氨基酸残基的取代。 R1057A 和 K1086A 蛋白不补充 rev3 Delta 突变，并且相对于野生型蛋白，这些蛋白的聚合酶活性显着降低。相比之下，K1061A 蛋白部分补充了 rev3 Delta 突变，并具有几乎正常的聚合酶活性。有趣的是，K1061A 蛋白相对于野生型 pol zeta 具有更高的保真度，并且从不匹配的引物末端碱基对延伸的效率稍低。这些发现对于 pol zeta 与经典聚合酶的进化分歧以及该酶适应由错配和损伤引起的 DNA 扭曲的机制具有重要意义。
DNA polymerase zeta (pol zeta), which is required for DNA damage-induced mutagenesis, functions in the error-prone replication of a wide range of DNA lesions. During this process, pol zeta extends from nucleotides incorporated opposite template lesions by other polymerases. Unlike classical polymerases, pol zeta efficiently extends from primer-terminal base pairs containing mismatches or lesions, and it synthesizes DNA with moderate fidelity. Here we describe genetic and biochemical studies of three yeast pol zeta mutant proteins containing substitutions of highly conserved amino acid residues that contact the triphosphate moiety of the incoming nucleotide. The R1057A and K1086A proteins do not complement the rev3 Delta mutation, and these proteins have significantly reduced polymerase activity relative to the wild-type protein. In contrast, the K1061A protein partially complements the rev3 Delta mutation and has nearly normal polymerase activity. Interestingly, the K1061A protein has increased fidelity relative to wild-type pol zeta and is somewhat less efficient at extending from mismatched primer-terminal base pairs. These findings have important implications both for the evolutionary divergence of pol zeta from classical polymerases and for the mechanism by which this enzyme accommodates distortions in the DNA caused by mismatches and lesions.