EndoMS is a recently identified mismatch specific endonuclease in Thermococcales of Archaea and Mycobacteria of Bacteria. The homologs of EndoMS are conserved in Archaea and Actinobacteria, where classic MutS-MutL-mediated DNA mismatch repair pathway is absent or non-functional. Here, we report a study on the in vitro mismatch cleavage activity and in vivo function of an EndoMS homolog (SisEndoMS) from Sulfolobus islandicus REY15A, the model archaeon belonging to Crenarchaeota. SisEndoMS is highly active on duplex DNA containing G/T, G/G, and T/T mismatches. Interestingly, the cleavage activity of SisEndoMS is stimulated by the heterotrimeric PCNAs, and when Mn²⁺ was used as the co-factor instead of Mg²⁺, SisEndoMS was also active on DNA substrates containing C/T or A/G mismatches, suggesting that the endonuclease activity can be regulated by ion co-factors and accessory proteins. We compared the spontaneous mutation rate of the wild type strain REY15A and ∆endoMS by counter selection against 5-fluoroorotic acid (5-FOA). The endoMS knockout mutant had much higher spontaneous mutation rate (5.06 × 10⁻³) than that of the wild type (4.6 × 10⁻⁶). A mutation accumulation analysis also showed that the deletion mutant had a higher mutation occurrence than the wild type, with transition mutation being the dominant, suggesting that SisEndoMS is responsible for mutation avoidance in this hyperthermophilic archaeon. Overexpression of the wild type SisEndoMS in S. islandicus resulted in retarded growth and abnormal cell morphology, similar to strains overexpressing Hje and Hjc, the Holliday junction endonucleases. Transcriptomic analysis revealed that SisEndoMS overexpression led to upregulation of distinct gene including the CRISPR-Cas IIIB system, methyltransferases, and glycosyltransferases, which are mainly localized to specific regions in the chromosome. Collectively, our results support that EndoMS proteins represent a noncanonical DNA repair pathway in Archaea. The mechanism of the mismatch repair pathway in Sulfolobus which have a single chromosome is discussed.

EndoMS是最近发现的古细菌嗜热球菌和细菌分枝杆菌的错配特异性核酸内切酶。EndoMS的同源物在古细菌和放线菌中是保守的，经典的MutS-MutL介导的DNA错配修复途径不存在或无功能。在这里，我们报告了一项针对体外 错配切割活性和 体内 EndoMS同源物（SisEndoMS）的功能 海岛REY15A，属于Crenarchaeota的原型古细菌。SisEndoMS在含有G / T，G / G和T / T错配的双链DNA上具有很高的活性。有趣的是，异三聚体PCNA刺激了SisEndoMS的切割活性，当使用Mn ²⁺代替Mg ²⁺作为辅因子时，SisEndoMS在含有C / T或A / G错配的DNA底物上也具有活性。核酸内切酶的活性可以通过离子辅因子和辅助蛋白来调节。我们比较了野生型REY15A和Δ的自发突变率内质通过针对5-氟乳清酸（5-FOA）的反选择。的内质剔除突变体的自发突变率（5.06×10 ^-3）比野生型（4.6×10 ^-6）高得多。突变积累分析还显示，与野生型相比，缺失突变体的突变发生率更高，而过渡突变是主要突变体，表明SisEndoMS负责该超嗜热古菌的突变避免。野生型SisEndoMS在大肠杆菌中过表达S. islandicus导致生长迟缓和异常细胞形态，类似于过表达Hje和Hjc（霍利迪连接核酸内切酶）的菌株。转录组学分析显示，SisEndoMS的过表达导致不同基因的上调，包括CRISPR-Cas IIIB系统，甲基转移酶和糖基转移酶，这些基因主要位于染色体的特定区域。总的来说，我们的结果支持EndoMS蛋白代表古细菌中的非经典DNA修复途径。错配修复途径的机制硫磺菌属 讨论具有单个染色体的基因。