Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family
Structure and function of the Escherichia coli RecE protein, a member of the RecB nuclease domain family
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DOI：
10.1074/jbc.m108627200
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发表时间：
2001-12-07
影响因子：
4.8
通讯作者：
Julin, DA
中科院分区：
文献类型：
--
作者：
Chang, HSW;Julin, DA
大肠杆菌 RecBCD 酶的 RecB 亚基具有解旋酶和核酸酶活性。解旋酶功能位于 N 端结构域，而核酸酶活性位于 C 端结构域。最近的分析发现了一组与 RecB 核酸酶结构域具有弱氨基酸序列相似性的蛋白质，并且被提议构成相关蛋白质家族（Axavind，L.，Walker，D.R.，和 Koonin，E.V.（1999）Nucleic Acids Res.27，1223-1242）。一种是大肠杆菌 RecE 蛋白（核酸外切酶 VIII），一种不依赖于 ATP 的核酸外切酶，可降解双链 DNA 的 5' 末端链。我们对 RecE 的几个残基进行了突变，与 RecB 的关键残基对齐，我们发现这些突变降低或消除了 RecE 的核酸酶活性，但不影响酶与线性双链 DNA 的结合。使用枯草杆菌蛋白酶的蛋白水解实验表明，包含这些关键残基的稳定的 34 千道尔顿 C 端结构域具有核酸酶活性，而 RecE 的 N 端部分没有积累稳定的蛋白水解片段。这些结果表明，RecE 具有与 RecB 相似的核酸酶结构域和活性位点，尽管两种蛋白质之间的序列相似性非常弱。这些相似性支持了两种蛋白质的核酸酶结构域在进化上相关的假设。
The RecB subunit of the Escherichia coli RecBCD enzyme has both helicase and nuclease activities. The helicase function was localized to an N-terminal domain, whereas the nuclease activity was found in a C-terminal domain. Recent analysis has uncovered a group of proteins that have weak amino acid sequence similarity to the RecB nuclease domain and that are proposed to constitute a family of related proteins (Axavind, L., Walker, D. R., and Koonin, E. V. (1999) Nucleic Acids Res. 27,1223-1242). One is the E. coli RecE protein (exonuclease VIII), an ATP-independent exonuclease that degrades the 5 ' -terminated strand of double-stranded DNA. We have made mutations in several residues of RecE that align with the critical residues of RecB, and we find that the mutations reduce or abolish the nuclease activity of RecE but do not affect the enzyme binding to linear double-stranded DNA. Proteolysis experiments with subtilisin show that a stable 34-kilodalton C-terminal domain that contains these critical residues has nuclease activity, whereas no stable proteolytic fragments accumulate from the N-terminal portion of RecE. These results show that RecE has a nuclease domain and active site that are similar to RecB, despite the very weak sequence similarity between the two proteins. These similarities support the hypothesis that the nuclease domains of the two proteins are evolutionarily related.