Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense
Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense
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DOI：
10.1016/j.str.2009.03.019
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发表时间：
2009-06-10
期刊：
影响因子：
5.7
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
--
作者：
Wiedenheft, Blake;Zhou, Kaihong;Doudna, Jennifer A.
原核生物中的获得性免疫是通过将外源核酸的短片段整合到成簇的规则间隔短回文重复序列（CRISPR）中来实现的。这种基于核酸的免疫系统由多达45个蛋白质家族的可变盒介导，这些蛋白质家族代表不同的免疫系统亚型。CRISPR相关基因1（cas1）编码CRISPR免疫系统中唯一普遍保守的蛋白质组分，但其功能尚不清楚。在这里，我们表明，Cas1蛋白是一种金属依赖的DNA特异性内切酶，产生类似于80个碱基对长度的双链DNA片段。Cas1蛋白的2.2埃晶体结构揭示了一个独特的折叠和一个保守的二价金属离子结合位点。金属离子结合残基的突变、金属离子的螯合或金属离子取代抑制Cas1催化的DNA降解。这些结果为理解Cas1如何促进CRISPR功能提供了基础，可能是处理外源核酸的机制的一部分。
Acquired immunity in prokaryotes is achieved by integrating short fragments of foreign nucleic acids into clustered regularly interspaced short palindromic repeats (CRISPRs). This nucleic acid-based immune system is mediated by a variable cassette of up to 45 protein families that represent distinct immune system subtypes. CRISPR-associated gene 1 (cas1) encodes the only universally conserved protein component of CRISPR immune systems, yet its function is unknown. Here we show that the Cas1 protein is a metal-dependent DNA-specific endonuclease that produces double-stranded DNA fragments of similar to 80 base pairs in length. The 2.2 angstrom crystal structure of the Cas1 protein reveals a distinct fold and a conserved divalent metal ion-binding site. Mutation of metal ion-binding residues, chelation of metal ions, or metal-ion substitution inhibits Cas1-catalyzed DNA degradation. These results provide a foundation for understanding how Cas1 contributes to CRISPR function, perhaps as part of the machinery for processing foreign nucleic acids.