Arslan Zihni, Hermanns Veronica, Wurm Reinhild, Wagner Rolf, Pul Ümit
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany.
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Universitätsstraße 1, 40225 Düsseldorf, Germany
Nucleic Acids Res. 2014 Jul;42(12):7884-93. doi: 10.1093/nar/gku510. Epub 2014 Jun 11.
The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.
CRISPR-Cas系统（成簇规律间隔短回文重复序列及其相关蛋白）对外源核酸的适应性依赖于通过未知机制将外源核酸衍生序列作为新的间隔序列插入CRISPR阵列。我们在大肠杆菌中鉴定并表征了间隔序列整合的中间状态，并在体内绘制了染色体CRISPR阵列上的整合位点。结果表明，新间隔序列的插入是通过在前导近端重复序列的两条链上进行位点特异性切口，呈交错方式，并伴随着重复链产生的5'端与进入的间隔序列的3'端连接。这种协同的切割-连接反应依赖于Cas1蛋白的金属结合中心，并且需要Cas2的存在。通过使用具有突变重复序列的质粒定位CRISPR阵列进行获取分析，我们证明第一个重复序列的一级序列对于CRISPR阵列的切割和新间隔DNA的连接至关重要。
