Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication
Investigating the role of the little finger domain of Y-family DNA polymerases in low fidelity synthesis and translesion replication
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DOI：
10.1074/jbc.m405249200
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发表时间：
2004-07-30
影响因子：
4.8
通讯作者：
Woodgate, R
中科院分区：
文献类型：
--
作者：
Boudsocq, F;Kokoska, RJ;Woodgate, R
Dpo 4和Dbh是Y家族聚合酶，其来源于Sulfolobaceae的两种密切相关的菌株。然而，非常令人惊讶的是，这两种聚合酶在体外表现出不同的酶性质。例如，Dpo 4可以复制通过各种DNA损伤，但Dbh这样做的效率要低得多。当复制未受损的DNA时，Dpo 4倾向于进行碱基对替换，而Dbh主要进行单碱基缺失。总的来说，这两种蛋白质有54%的相同性，但最大的分歧是在它们各自的小指（LF）结构域，只有41%的相同性。为了研究LF结构域在Y家族聚合酶的保真度和病变绕过能力中的作用，我们产生了Dpo 4和Dbh的嵌合体，其中它们的LF结构域已经互换。有趣的是，通过用Dpo 4的LF结构域替换Dbh的LF结构域，嵌合酶的酶性质更像Dpo 4，因为该酶更具有进行性，可以绕过脱碱基位点和胸腺嘧啶-胸腺嘧啶环丁烷嘧啶二聚体，并且在复制未受损的DNA时主要进行碱基对取代。匡威的是，Dpo 4-LF-Dbh嵌合体，这是更像Dbh的合成能力和能力，绕过DNA加合物和产生单碱基缺失错误。我们的研究表明，独特的，但可变的LF结构域的Y-家族聚合酶起着重要的作用，在确定每个人的Y-家族成员的酶和生物学特性。
Dpo4 and Dbh are Y-family polymerases that originate from two closely related strains of Sulfolobaceae. Quite surprisingly, however, the two polymerases exhibit different enzymatic properties in vitro. For example, Dpo4 can replicate past a variety of DNA lesions, yet Dbh does so with a much lower efficiency. When replicating undamaged DNA, Dpo4 is prone to make base pair substitutions, whereas Dbh predominantly makes single-base deletions. Overall, the two proteins are 54% identical, but the greatest divergence is found in their respective little finger (LF) domains, which are only 41% identical. To investigate the role of the LF domain in the fidelity and lesion-bypassing abilities of Y-family polymerases, we have generated chimeras of Dpo4 and Dbh in which their LF domains have been interchanged. Interestingly, by replacing the LF domain of Dbh with that of Dpo4, the enzymatic properties of the chimeric enzyme are more Dpo4-like in that the enzyme is more processive, can bypass an abasic site and a thymine-thymine cyclobutane pyrimidine dimer, and predominantly makes base pair substitutions when replicating undamaged DNA. The converse is true for the Dpo4-LF-Dbh chimera, which is more Dbh-like in its processivity and ability to bypass DNA adducts and generate single-base deletion errors. Our studies indicate that the unique but variable LF domain of Y-family polymerases plays a major role in determining the enzymatic and biological properties of each individual Y-family member.