Laboratory of Molecular Genetics, NIEHS, National Institutes of Health, Research Triangle Park, NC 27709, United States.
DNA Repair (Amst). 2010 Apr 4;9(4):448-57. doi: 10.1016/j.dnarep.2010.01.010.
Saccharomyces cerevisiae MutLalpha is a heterodimer of Mlh1 and Pms1 that participates in DNA mismatch repair (MMR). Both proteins have weakly conserved C-terminal regions (CTDs), with the CTD of Pms1 harboring an essential endonuclease activity. These proteins also have conserved N-terminal domains (NTDs) that bind and hydrolyze ATP and bind to DNA. To better understand Pms1 functions and potential interactions with DNA and/or other proteins, we solved the 2.5A crystal structure of yeast Pms1 (yPms1) NTD. The structure is similar to the homologous NTDs of Escherichia coli MutL and human PMS2, including the site involved in ATP binding and hydrolysis. The structure reveals a number of conserved, positively charged surface residues that do not interact with other residues in the NTD and are therefore candidates for interactions with DNA, with the CTD and/or with other proteins. When these were replaced with glutamate, several replacements resulted in yeast strains with elevated mutation rates. Two replacements also resulted in NTDs with decreased DNA binding affinity in vitro, suggesting that these residues contribute to DNA binding that is important for mismatch repair. Elevated mutation rates also resulted from surface residue replacements that did not affect DNA binding, suggesting that these conserved residues serve other functions, possibly involving interactions with other MMR proteins.
酿酒酵母 MutLalpha 是由 Mlh1 和 Pms1 组成的异二聚体，参与 DNA 错配修复 (MMR)。这两种蛋白质都有较弱保守的 C 端区域 (CTD)，其中 Pms1 的 CTD 具有必需的内切核酸酶活性。这些蛋白质还具有保守的 N 端结构域 (NTD)，可结合和水解 ATP，并与 DNA 结合。为了更好地理解 Pms1 的功能以及与 DNA 和/或其他蛋白质的潜在相互作用，我们解析了酿酒酵母 Pms1 (yPms1) NTD 的 2.5A 晶体结构。该结构与大肠杆菌 MutL 和人 PMS2 的同源 NTD 相似，包括参与 ATP 结合和水解的位点。该结构揭示了许多保守的、带正电荷的表面残基，这些残基不与 NTD 中的其他残基相互作用，因此是与 DNA、CTD 和/或其他蛋白质相互作用的候选者。当这些残基被谷氨酸取代时，几个取代导致酵母菌株的突变率升高。两个替换物也导致 NTD 在体外的 DNA 结合亲和力降低，表明这些残基有助于对错配修复很重要的 DNA 结合。表面残基取代物不影响 DNA 结合也会导致突变率升高，这表明这些保守残基具有其他功能，可能涉及与其他 MMR 蛋白的相互作用。
