Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A.
Functional Analysis of the NucS/EndoMS of the Hyperthermophilic Archaeon Sulfolobus islandicus REY15A.
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DOI：
10.3389/fmicb.2020.607431
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发表时间：
2020
影响因子：
5.2
通讯作者：
Shen Y
中科院分区：
文献类型：
--
作者：
Ahmad S;Huang Q;Ni J;Xiao Y;Yang Y;Shen Y
EndoMS是最近在细菌的分支杆菌和热球菌中发现的一种错配特异性内切酶。EndoMS的同源物在放线菌和放线菌中是保守的，其中经典的MutS-MutL介导的DNA错配修复途径不存在或无功能。在这里，我们报告了一个EndoMS同源物（SisEndoMS）的体外错配切割活性和体内功能的研究，从硫化叶菌islandicus REY 15 A，模式古菌属于Crenarchaeota。SisEndoMS对含有G/T、G/G和T/T错配的双链体DNA具有高度活性。有趣的是，SisEndoMS的切割活性受到异源三聚体PCNAs的刺激，并且当Mn 2+代替Mg 2+用作辅因子时，SisEndoMS也对含有C/T或A/G错配的DNA底物具有活性，这表明内切核酸酶活性可以通过离子辅因子和辅助蛋白来调节。我们通过对5-氟乳清酸（5-FOA）的反选择比较了野生型菌株REY 15 A和EscherichiMS的自发突变率。endoMS敲除突变体的自发突变率（5.06 × 10−3）远高于野生型（4.6 × 10−6）。突变累积分析还表明，缺失突变体比野生型具有更高的突变发生率，过渡突变占主导地位，这表明SisEndoMS负责在这种极端嗜热古菌中避免突变。野生型SisEndoMS在S. islandicus导致生长迟缓和细胞形态异常，类似于过表达Hje和Hjc（Holliday连接核酸内切酶）的菌株。转录组学分析显示，SisEndoMS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，这些基因主要定位于染色体中的特定区域。总的来说，我们的研究结果支持EndoMS蛋白代表了一个非经典的DNA修复途径在大肠杆菌。对单染色体硫化叶菌的错配修复机制进行了讨论。
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.
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