Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

译文

聚集的规则间隔的短回文重复序列 (CRISPRs) 和相关蛋白 (Cas) 构成了针对原核生物中病毒和质粒的适应性免疫系统。Cas1是一种与CRISPR相关的蛋白质,对所有含CRISPR的原核生物都很常见,但其功能仍然不清楚。在这里,我们显示了大肠杆菌 (YgbT) 的纯化Cas1蛋白对单链和分支dna (包括Holliday连接,复制叉和5'-皮瓣) 具有核酸酶活性。YgbT的晶体结构和定点诱变揭示了潜在的活性位点。全基因组屏幕显示,YgbT与DNA修复系统的关键组件 (包括recB,recC和ruvB) 在物理和遗传上相互作用。与这些发现一致,ygbT缺失菌株显示出对DNA损伤的敏感性增加,染色体分离受损。在CRISPR簇缺失的菌株中观察到类似的表型,表明YgbT在修复中的功能涉及与CRISPR的相互作用。这些结果表明,YgbT属于作用于分支DNA的新型,结构上不同的核酸酶家族,并表明,除了抗病毒免疫外,CRISPR-Cas系统的至少某些组件在DNA修复中具有功能。

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