古菌及放线菌大都缺乏经典的依赖MutS/MutL的错配修复途径，其错配修复方式一直是个未解之谜。最近在嗜热广古菌和放线菌中鉴定到一种偏好性地识别G/G、G/T和T/T错配的核酸内切酶，命名为EndoMS/NucS, 可能介导一种非典型的错配修复途径。然而，其活性在古菌中的普遍性、体内功能和作用机制未知。本项目以模式超嗜热泉古菌冰岛硫化叶菌(Sulfolobus islandicus REY15A)为材料，采用生物化学和遗传学方法，分析泉古菌中EndoMS的生化性质、基因必需性、基因缺失以及蛋白超表达对菌株生长、损伤剂敏感性和基因突变谱的影响；筛选并鉴定与EndoMS互作、或者识别其它种类错配底物的蛋白或酶; 利用定向进化方法，筛选专一性地识别特定错配的EndoMS突变蛋白。研究结果将有助于全面理解EndonMS性质，拓展酶的应用，揭示EndoMS介导的错配修复途径的普遍性、特征和作用机制。

Almost all of Archaea and actinomycetes lack canonical MutS/MutL-dependent mismatch repair pathway. The way for its mismatch repair has long been a mystery. Recently, an endonuclease, named EndoMS, was identified in some thermophile Euryarchaeota and actinomycetes. It specifically recognizes and cleavages DNA with G/G，G/T，and T/T mismatches, presumably mediating a non-canonical mismatch repair pathway. However, its universality, in vivo function, and mechanism remain unclear. In this study, using Sulfolobusislandicus REY15A as a model crenarchaeon and taking biochemical and genetic approaches, we will analyze the biochemical properties of EndoMS in Crenarcheota, the gene essentiality, the effects of gene knockout, protein overexpression on the growth, and sensitivity to DNA damage agents, as well as mutation profile of the cells. We will also identify putative proteins that can recognize other types of mismatches or interact with EndoMS. At the same time, we will use directed mutagenesis to screen out mutants that can specifically recognize DNA containing a single mismatch. The results will help comprehensively understand properties of EndoMS, expand its application, and will reveal the universality of the activity and unveil the features and mechanism of this novel EndoMS mediated mismatch repair pathway.