Crystal Structure of the Csm1 Subunit of the Csm Complex and Its Single-Stranded DNA-Specific Nuclease Activity
Crystal Structure of the Csm1 Subunit of the Csm Complex and Its Single-Stranded DNA-Specific Nuclease Activity
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DOI：
10.1016/j.str.2015.01.021
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发表时间：
2015-04-07
期刊：
影响因子：
5.7
通讯作者：
Woo, Euijeon
中科院分区：
文献类型：
--
作者：
Jung, Tae-Yang;An, Yan;Woo, Euijeon
CRISPR-Cas系统是细菌和古细菌中RNA引导的免疫防御机制。Csm 1属于Cas 10家族，其是III型系统的共同特征蛋白。Csm 1是Csm干扰复合物中最大的亚基，靶向外源DNA或RNA。在这里，我们报告的晶体学和生化分析嗜热球菌CSM 1，揭示了五个结构域的组织和单链DNA（ssDNA）的特异性核酸酶活性与N-末端HD域。与Cas 3的HD结构域相比，该结构域折叠成排列的二级结构，并且包含所有催化重要的残基。它以Ni ~（2+）或Mn ~（2+）依赖的方式表现出内切和外切核酸酶活性。活性位点裂缝的狭窄宽度似乎限制了底物对ssDNA的特异性，从而防止Csm 1切割双链染色体DNA。这些数据表明，CSM 1可能在DNA干扰的CSM效应复合物的功能。
The CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo-and exonuclease activities in an Ni2+ or Mn2+-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.