The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair.
The unstructured linker arms of Mlh1-Pms1 are important for interactions with DNA during mismatch repair.
复制标题
Mlh1-Pms1 的非结构化连接臂对于错配修复过程中与 DNA 的相互作用非常重要。
DOI：
10.1016/j.jmb.2012.05.030
复制
发表时间：
2012
影响因子：
5.6
通讯作者：
Alani,Eric
中科院分区：
文献类型：
--
作者：
Plys,AaronJ;Rogacheva,MariaV;Greene,EricC;Alani,Eric
DNA错配修复（MMR）模型提出MSH（MutS同系物）蛋白在与DNA复制叉相互作用时识别DNA聚合酶错误。MLH（MutL同源物）蛋白（主要是面包酵母中的Mlh 1-Pms 1）然后调查基因组中的损伤结合的MSH蛋白。在DNA损伤处形成的MSH-MLH复合物启动修复的下游步骤。MLH蛋白作为二聚体，含有连接两个末端球状结构域的长（20- 30 nm）非结构化臂。这些臂的长度可以在100至300个氨基酸之间变化，在生物体之间高度不同，并且对氨基酸取代具有抗性。为了测试连接臂在MMR中的作用，我们将蛋白酶切割位点工程化到面包酵母Mlh 1-Pms 1的Mlh 1连接臂结构域中。Mlh 1连接臂的体外切割导致Mlh 1-Pms 1 DNA结合活性的缺陷，而体内蛋白水解切割导致MMR的完全缺陷。然后，我们产生了一系列截短突变体轴承Mlh 1和Pms 1连接臂的不同长度。这项工作表明，MMR是大大损害时，部分的Mlh 1连接器被删除，而修复是不太敏感的截断的Pms 1连接器arm. Purified复合物中含有截断Mlh 1和Pms 1连接器臂进行了分析，发现有差异的缺陷，DNA结合，也与形成一个三元复合物的能力与Msh 2-Msh 6和错配DNA。这些观察结果与MLH蛋白的非结构化接头结构域在MMR期间提供与DNA的不同相互作用一致。
DNA mismatch repair (MMR) models have proposed that MSH (MutS homolog) proteins identify DNA polymerase errors while interacting with the DNA replication fork. MLH (MutL homolog) proteins (primarily Mlh1–Pms1 in baker's yeast) then survey the genome for lesion-bound MSH proteins. The resulting MSH–MLH complex formed at a DNA lesion initiates downstream steps in repair. MLH proteins act as dimers and contain long (20–30nm) unstructured arms that connect two terminal globular domains. These arms can vary between 100 and 300 amino acids in length, are highly divergent between organisms, and are resistant to amino acid substitutions. To test the roles of the linker arms in MMR, we engineered a protease cleavage site into the Mlh1 linker arm domain of baker's yeast Mlh1–Pms1. Cleavage of the Mlh1 linker arm in vitro resulted in a defect in Mlh1–Pms1 DNA binding activity, and in vivo proteolytic cleavage resulted in a complete defect in MMR. We then generated a series of truncation mutants bearing Mlh1 and Pms1 linker arms of varying lengths. This work revealed that MMR is greatly compromised when portions of the Mlh1 linker are removed, whereas repair is less sensitive to truncation of the Pms1 linker arm. Purified complexes containing truncations in Mlh1 and Pms1 linker arms were analyzed and found to have differential defects in DNA binding that also correlated with the ability to form a ternary complex with Msh2–Msh6 and mismatch DNA. These observations are consistent with the unstructured linker domains of MLH proteins providing distinct interactions with DNA during MMR.