Mutations in human DNA polymerase η motif II alter bypass of DNA lesions
Mutations in human DNA polymerase η motif II alter bypass of DNA lesions
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DOI：
10.1093/emboj/20.24.7303
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发表时间：
2001-12-17
期刊：
影响因子：
11.4
通讯作者：
Loeb, LA
中科院分区：
文献类型：
--
作者：
Glick, E;Vigna, KL;Loeb, LA
人DNA聚合酶ETA(HPOL ETA)是新发现的Y-家族DNA聚合酶之一。这些聚合酶合成过去的模板损伤，这些损伤被认为可以阻止复制分叉进展。HPOL ETA在体外准确地绕过紫外线相关的顺式环丁烷胸腺嘧啶二聚体，并有助于正常抵抗阳光诱导的皮肤癌。我们在这里描述了对Motif II的突变分析，Motif II是一个高度保守的序列，最近被报道位于Finger结构域，并形成Y家族DNA聚合酶活性部位的一部分。我们使用基于酵母的互补系统来分离通过随机序列突变产生的具有生物活性的突变株，在体外合成突变蛋白，并评估它们绕过胸腺嘧啶二聚体的能力。在210个活性突变体中，基序II的可变性与自然进化相似，并确定Tyr52和Ala54是参与催化活性或旁路的主要候选者。我们描述了HPOL ETA S62G，一种活性增强的突变聚合酶，绕过其他五个特定部位病变的能力。我们的结果可能为研究Y家族DNA聚合酶的其他成员提供一个原型。
Human DNA polymerase eta (hPol eta) is one of the newly identified Y-family of DNA polymerases. These polymerases synthesize past template lesions that are postulated to block replication fork progression. hPol eta accurately bypasses UV-associated cis-syn cyclobutane thymine dimers in vitro and contributes to normal resistance to sunlight-induced skin cancer. We describe here mutational analysis of motif II, a highly conserved sequence, recently reported to reside in the fingers domain and to form part of the active site in Y-family DNA polymerases. We used a yeast-based complementation system to isolate biologically active mutants created by random sequence mutagenesis, synthesized the mutant proteins in vitro and assessed their ability to bypass thymine dimers. The mutability of motif II in 210 active mutants has parallels with natural evolution and identifies Tyr52 and Ala54 as prime candidates for involvement in catalytic activity or bypass. We describe the ability of hPol eta S62G, a mutant polymerase with enhanced activity, to bypass five other site-specific lesions. Our results may serve as a prototype for studying other members of the Y-family DNA polymerases.