Department of Chemical Engineering and Applied Chemistry, Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
EMBO J. 2011 Oct 18;30(22):4616-27. doi: 10.1038/emboj.2011.377.
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') single-stranded 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.
成簇规律间隔短回文重复序列（CRISPRs）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防御系统。Cas3 蛋白被认为在 CRISPR 机制中通过对入侵 DNA 的直接切割发挥关键作用。在这里，我们表明，产甲烷球菌（Methanocaldococcus jannaschii）的 Cas3 HD 结构域蛋白 MJ0384 可以对单链 DNA 和 RNA 进行内切和外切（3'-5'）切割，以及 3'-发夹、展开臂和 R 环。Cas3 解旋酶 MJ0383 和 ATP 可刺激 MJ0384 对分支 DNA 底物的降解。MJ0384 的晶体结构揭示了具有两个结合金属阳离子的活性位点，与定点突变实验一起提出了一种催化机制。我们的研究表明，Cas3 HD 核酸酶与 Cas3 解旋酶一起工作，通过内切酶和外切酶活性的组合，可以完全降解入侵 DNA。
