In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination
In vitro and in vivo studies of MutS, MutL and MutH mutants: correlation of mismatch repair and DNA recombination
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DOI：
10.1016/s1568-7864(02)00245-8
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发表时间：
2003-04-02
期刊：
影响因子：
3.8
通讯作者：
Miller, JH
中科院分区：
文献类型：
--
作者：
Junop, MS;Yang, W;Miller, JH
我们利用最近确定的大肠杆菌MutS、MutL和MuTH的晶体结构来指导这些蛋白质中47个氨基酸取代位的构建，并分析了它们在体内外错配修复和重组中的行为。我们发现，Muth内切酶的活性部位由来自两个独立结构域的区域组成，Muth的C-末端5个残基既影响DNA的结合，也影响DNA的切割。我们还发现MutL的非特异性DNA结合活性是错配修复所必需的，并可能在Muth切割链后发挥作用。错配识别结构域、ATPase活性位点或连接这两个活性的结构域界面上的残基的改变可以降低MutS与同源双链和异源双链的差异结合，并导致错配特异性MuTH激活的丧失。最后，每个取消错配修复的突变都缺乏阻止同源重组的能力，这表明错配修复和阻止同源重组在大肠杆菌中使用相同的MutS-MutL复合体来传递信号。爱思唯尔科学公司出版。
We have used the recently determined crystal structures of Escherichia coli (E. coli) MutS, MutL and MutH to guide construction of 47 amino-acid substitutions in these proteins and analyzed their behavior in mismatch repair and recombination in vitro and in vivo. We find that the active site of the MutH endonuclease is composed of regions from two separate structural domains and that the C-terminal 5 residues of MutH influence both DNA binding and cleavage. We also find that the non-specific DNA-binding activity of MutL is required for mismatch repair and probably functions after strand cleavage by MutH. Alteration of residues in either the mismatch recognition domain, the ATPase active site, or the domain interfaces linking the two activities can diminish the differential binding of MutS to homoduplex versus heteroduplex and results in the loss of mismatch-specific MutH activation. Finally, every mutation that abolishes mismatch repair is deficient in blocking homeologous recombination, suggesting that mismatch repair and prevention of homeologous recombination use the same MutS-MutL complexes for signaling in E. coli. Published by Elsevier Science B.V.