CRISPR adaptation biases explain preference for acquisition of foreign DNA.
CRISPR adaptation biases explain preference for acquisition of foreign DNA.
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DOI：
10.1038/nature14302
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发表时间：
2015-04-23
期刊：
影响因子：
64.8
通讯作者：
Sorek R
中科院分区：
文献类型：
--
作者：
Levy A;Goren MG;Yosef I;Auster O;Manor M;Amitai G;Edgar R;Qimron U;Sorek R
在 CRISPR 适应过程中，从外来元件中获取短 DNA 片段（“间隔区”）并整合到 CRISPR 阵列中。迄今为止，如何优先从外源 DNA 中获得间隔区，同时避免自体染色体仍然是一个谜。在这里，我们表明间隔区的获取是复制依赖性的，并且在停滞的复制叉处形成的DNA断裂促进间隔区的获取。间隔区获取的染色体热点受到 Chi 位点的限制，Chi 位点是细菌染色体上高度富集的序列八聚体，表明这些位点限制了从自身 DNA 获取间隔区。我们进一步表明，对“自我”的回避是由 RecBCD dsDNA 断裂修复复合物介导的。我们的结果表明，在大肠杆菌中，新间隔区的获得取决于 RecBCD 介导的主要发生在复制叉处的 dsDNA 断裂处理，并且对外来 DNA 的偏好是通过自身染色体上较高密度的 Chi 位点以及外来 DNA 上较多的叉子来实现的。该模型解释了从高拷贝质粒和噬菌体中获取间隔区的强烈偏好。
In the process of CRISPR adaptation, short pieces of DNA (“spacers”) are acquired from foreign elements and integrated into the CRISPR array. It so far remained a mystery how spacers are preferentially acquired from the foreign DNA while the self chromosome is avoided. Here we show that spacer acquisition is replication-dependent, and that DNA breaks formed at stalled replication forks promote spacer acquisition. Chromosomal hotspots of spacer acquisition were confined by Chi sites, which are sequence octamers highly enriched on the bacterial chromosome, suggesting that these sites limit spacer acquisition from self DNA. We further show that the avoidance of “self” is mediated by the RecBCD dsDNA break repair complex. Our results suggest that in E. coli, acquisition of new spacers depends on RecBCD-mediated processing of dsDNA breaks occurring primarily at replication forks, and that the preference for foreign DNA is achieved through the higher density of Chi sites on the self chromosome, in combination with the higher number of forks on the foreign DNA. This model explains the strong preference to acquire spacers from both high copy plasmids and phages.