Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.
Department of Chemistry, University of California, Berkeley, Berkeley, California, USA.
Nat Struct Mol Biol. 2016 Oct;23(10):876-883. doi: 10.1038/nsmb.3289. Epub 2016 Sep 5.
Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1-Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.
细菌 CRISPR-Cas 系统包括侧翼带有外源 DNA 序列的短重复基因组阵列，并提供针对病毒的适应性免疫。为了避免损害基因组或 CRISPR 阵列，外源 DNA 的整合必须是特异性的，但令人惊讶的是，体外会发生杂乱无章的活性。在这里，我们重新构建了完整位点的 DNA 整合，并表明链球菌 II-A Cas1-Cas2 整合酶通过限制第二步整合在一定程度上保持特异性。在非 CRISPR 位点，整合在半位点中间体处停滞，从而使反应能够逆转。S. pyogenes Cas1-Cas2 高度特异性识别先导近端重复序列，并识别重复序列的回文末端，因此符合远端 Cas1 活性位点独立识别的模型。这些发现表明，DNA 插入位点比以前的工作所表明的要少，从而防止了 CRISPR 免疫适应过程中的毒性，并保持了宿主基因组的完整性。
