Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Chemistry, University of California, Berkeley, Berkeley, CA 94720, USA.
Mol Cell. 2019 Feb 21;73(4):727-737.e3. doi: 10.1016/j.molcel.2018.12.015. Epub 2019 Jan 29.
CRISPR-Cas immunity requires integration of short, foreign DNA fragments into the host genome at the CRISPR locus, a site consisting of alternating repeat sequences and foreign-derived spacers. In most CRISPR systems, the proteins Cas1 and Cas2 form the integration complex and are both essential for DNA acquisition. Most type V-C and V-D systems lack the cas2 gene and have unusually short CRISPR repeats and spacers. Here, we show that a mini-integrase comprising the type V-C Cas1 protein alone catalyzes DNA integration with a preference for short (17- to 19-base-pair) DNA fragments. The mini-integrase has weak specificity for the CRISPR array. We present evidence that the Cas1 proteins form a tetramer for integration. Our findings support a model of a minimal integrase with an internal ruler mechanism that favors shorter repeats and spacers. This minimal integrase may represent the function of the ancestral Cas1 prior to Cas2 adoption.
CRISPR-Cas 免疫需要将短的外源 DNA 片段整合到 CRISPR 基因座的宿主基因组中，该基因座由交替重复序列和外源衍生的间隔区组成。在大多数 CRISPR 系统中，Cas1 和 Cas2 蛋白形成整合复合物，两者对于 DNA 的获取都是必需的。大多数 V-C 和 V-D 型系统缺乏 cas2 基因，并且 CRISPR 重复序列和间隔区非常短。在这里，我们表明，一个由 V-C 型 Cas1 蛋白单独组成的小型整合酶可催化 DNA 整合，偏爱短（17 至 19 碱基对）的 DNA 片段。该小型整合酶对 CRISPR 序列的特异性较弱。我们提供的证据表明，Cas1 蛋白形成四聚体以进行整合。我们的研究结果支持一种最小整合酶的模型，该模型具有内部标尺机制，有利于较短的重复序列和间隔区。这种最小整合酶可能代表了 Cas2 采用之前 Cas1 蛋白的祖先功能。
