Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity.
Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity.
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DOI：
10.1093/nar/gkv1213
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发表时间：
2015-12-15
影响因子：
14.9
通讯作者：
Bolt EL
中科院分区：
文献类型：
--
作者：
Ivančić-Baće I;Cass SD;Wearne SJ;Bolt EL
CRISPR-Cas是一种原核免疫系统，通过捕获入侵DNA并将其整合到CRISPR（规则间隔短回文重复序列）基因座中，称为“适应”，其依赖于Cas 1和Cas 2蛋白。在大肠杆菌中，Cascade-Cas 3降解入侵者DNA以影响免疫力，称为“干扰”。适应可以与干扰相互作用（“启动”），也可以独立于干扰（“幼稚”）。我们证明，引发的适应需要RecG解旋酶和PriA蛋白存在。突变体表型的遗传分析表明，需要RecG在阻断的复制叉处消散R环。此外，我们确定DNA聚合酶I对启动适应和幼稚适应都很重要，而RecB是幼稚适应所需的。纯化的Cas 1-Cas 2蛋白显示出特异性结合并切割单链间隙内的叉状DNA，并将叉折叠成DNA双链体。这些数据表明，不同的基因组稳定性系统与启动或幼稚适应时，响应阻断或崩溃的入侵者DNA复制。在该模型中，RecG和Cas 3蛋白响应于被Cascade干扰阻断的入侵者DNA复制叉，从而实现DNA捕获。RecBCD靶向折叠叉处的DNA末端，使DNA捕获不受干扰。DNA聚合酶I被认为是在间隔区整合过程中填补DNA缺口的酶。
CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.