CRISPR Immunological Memory Requires a Host Factor for Specificity
CRISPR Immunological Memory Requires a Host Factor for Specificity
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DOI：
10.1016/j.molcel.2016.04.027
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发表时间：
2016-06-16
期刊：
影响因子：
16
通讯作者：
Doudna, Jennifer A.
中科院分区：
文献类型：
--
作者：
Nunez, James K.;Bai, Lawrence;Doudna, Jennifer A.
细菌和古细菌使用CRISPR-Cas系统对外来遗传元件进行适应性免疫。为了产生免疫记忆，Cas 1-Cas 2蛋白复合物捕获外源DNA的30-40个碱基对片段，并催化它们作为独特的间隔序列整合到宿主基因组中。虽然间隔区在体内被严格插入在CRISPR基因座的富含A-T的前导序列末端，但前导序列特异性间隔区整合的分子机制仍然知之甚少。在这里，我们表明，E。大肠杆菌整合宿主因子（IHF）蛋白是体内获得间隔区和体外整合到线性DNA中所必需的。IHF与前导序列结合并诱导尖锐的DNA弯曲，使Cas 1Cas 2整合酶催化前导序列-重复序列边界处的第一次整合反应。总之，这些结果揭示了Cas 1-Cas 2介导的间隔区整合需要IHF诱导的靶DNA弯曲，并解释了CRISPR前导序列在间隔区获取过程中的难以捉摸的作用。
Bacteria and archaea employ adaptive immunity against foreign genetic elements using CRISPR-Cas systems. To generate immunological memory, the Cas1-Cas2 protein complex captures 30-40 base pair segments of foreign DNA and catalyzes their integration into the host genome as unique spacer sequences. Although spacers are inserted strictly at the A-T-rich leader end of CRISPR loci in vivo, the molecular mechanism of leader-specific spacer integration remains poorly understood. Here we show that the E. coli integration host factor (IHF) protein is required for spacer acquisition in vivo and for integration into linear DNA in vitro. IHF binds to the leader sequence and induces a sharp DNA bend, allowing the Cas1Cas2 integrase to catalyze the first integration reaction at the leader-repeat border. Together, these results reveal that Cas1-Cas2-mediated spacer integration requires IHF-induced target DNA bending and explain the elusive role of CRISPR leader sequences during spacer acquisition.