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Abstract：
CRISPR-Cas systems provide bacteria with adaptive immunity against viruses. During spacer adaptation, the Cas1-Cas2 complex selects fragments of foreign DNA, called prespacers, and integrates them into CRISPR arrays in an orientation that provides functional immunity. Cas4 is involved in both the trimming of prespacers and the cleavage of protospacer adjacent motif (PAM) in several type I CRISPR-Cas systems, but how the prespacers are processed in systems lacking Cas4, such as the type I-E and I-F systems, is not understood. In Escherichia coli, which has a type I-E system, Cas1-Cas2 preferentially selects prespacers with 3\' overhangs via specific recognition of a PAM, but how these prespacers are integrated in a functional orientation in the absence of Cas4 is not known. Using a biochemical approach with purified proteins, as well as integration, prespacer protection, sequencing, and quantitative PCR assays, we show here that the bacterial 3\'-5\' exonucleases DnaQ and ExoT can trim long 3\' overhangs of prespacers and promote integration in the correct orientation. We found that trimming by these exonucleases results in an asymmetric intermediate, because Cas1-Cas2 protects the PAM sequence, which helps to define spacer orientation. Our findings implicate the E. coli host 3\'-5\' exonucleases DnaQ and ExoT in spacer adaptation and reveal a mechanism by which spacer orientation is defined in E. coli.
摘要：
CRISPR-Cas系统为细菌提供对病毒的适应性免疫。在隔片自适应期间，Cas1-Cas2复合物选择外源DNA的片段，叫做预起搏器，并将它们以提供功能性免疫的方向整合到CRISPR阵列中。在几种I型CRISPR-Cas系统中，Cas4参与预缩放体的修剪和前间隔区相邻基序（PAM）的裂解，但是在缺乏Cas4的系统中如何处理预起搏器，例如I-E和I-F型系统，不理解。在大肠杆菌中，它有一个I-E型系统，Cas1-Cas2通过对PAM的特定识别，优先选择具有3''悬垂的预封装器，但是在不存在Cas4的情况下，这些预起搏器是如何以功能取向整合的，目前尚不清楚。使用纯化蛋白质的生化方法，以及整合，预起搏器保护,测序,测序和定量PCR检测，我们在这里表明，细菌3'-5'核酸外切酶DnaQ和ExoT可以修剪长长的3'突出部分，并促进正确方向的整合。我们发现通过这些外切核酸酶的修剪会导致不对称的中间体，因为Cas1-Cas2保护PAM序列,这有助于定义间隔物方向。我们的发现暗示大肠杆菌宿主3'-5'核酸外切酶DnaQ和ExoT参与间隔区适应，并揭示了在大肠杆菌中定义间隔区方向的机制。
