Structural and functional studies on Cas10, the putative catalytic subunit of the Csm complex in the CRISPR-Cas bacterial immune system

Abstract

The Csm complex is a crRNA-guided effector complex that cleaves invader DNA in the prokaryotic immune response mediated by the type III-A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)-Cas system. To understand function and mechanism of the crRNA-guided effector complex, I thoroughly examined CRISPR locus of hyperthermophilic archeon Thermococcus onnurineus and found the putative seven genes encoding Cas6, Csm1 (Cas10), Csm2, Csm3, Csm4, Csm5, and Csx1 in a row. They are expected to assemble Csm complex that involve in pre crRNA processing, crRNA maturation, and target DNA interference. Based on structural and biochemical studies, I determined the structure of Cas10 from T. onnurineus (Ton_Cas10) and demonstrated that Ton_Cas10 possesses metal-dependent (Mg, Mn) nuclease activity against single-strnaded (ss-)DNA, double-stranded (ds-)DNA and (ss-)RNA, proposing Cas10 to be the catalytic subunit of the complex. Structure revealed the overall dimeric arrangement of Cas10 with four domains in each monomer including the putative DNA Hydrolase Domain (HD). The detailed structure of HD domain showed motif II to V, which were highly conserved in classical HD domain. However, instead of motif I in other subfamily, Cas10 HD has H201 residue on helix 8, suggesting the two-metal nuclease system. The two similar ferrodoxin-like domains, D1 and D2, were analyzed and novel β-sheet appendix architecture was extended after Zn finger motif in D1 domain. It also shares structural homology with antiparallel β-strand structure similar to RRM (RNA Recognition Motif) fold. On D2 domain, conserved GGDDF motif structure was found and it suggests the ATPase activity function. According to structural analysis, I carried out the ATPase activity assay and found that Cas10 showed the ss- DNA stimulated ATPase activity with a possible helicase function. Based on these results, I conclude that Cas10 is the catalytic subunit of Csm complex in type III-A CRISPR system. Csm ...