Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference
Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference
复制标题
DOI：
10.1038/emboj.2011.377
复制
发表时间：
2011-11-16
期刊：
影响因子：
11.4
通讯作者：
Yakunin, Alexander F.
中科院分区：
文献类型：
--
作者：
Beloglazova, Natalia;Petit, Pierre;Yakunin, Alexander F.
簇状规则间隔短回文重复序列(CRISPR)和Cas蛋白代表了一种针对病毒和质粒的适应性微生物免疫系统。Cas3蛋白被认为通过直接切割侵袭性DNA在CRISPR机制中发挥关键作用。在这里，我们发现来自詹纳斯基甲烷球菌的Cas3 HD结构域蛋白MJ0384可以内切和外切(3‘-5’)单链DNA和RNA，以及3‘-翻盖、张开的手臂和R-环。MJ0384对分支DNA底物的降解受Cas3解旋酶MJ0383和三磷酸腺苷的刺激。MJ0384的晶体结构揭示了活性中心与两个结合的金属阳离子的结合，并结合定点突变提出了催化机制。我们的研究表明，Cas3 HD核酸酶与Cas3解旋酶一起工作，可以通过内切酶和外切酶的结合来完全降解侵袭性DNA。EMBO期刊(2011)30,4616-4627。DOI：10.1038/Intemj.2011.377；2011年10月18日在线发布
Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') singlestranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities. The EMBO Journal (2011) 30, 4616-4627. doi:10.1038/emboj.2011.377; Published online 18 October 2011