Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination
Mismatch sensing by nucleofilament deciphers mechanism of RecA-mediated homologous recombination
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通过核丝错配传感破译 RecA 介导的同源重组机制
DOI：
10.1073/pnas.1920265117
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发表时间：
2020-08-25
影响因子：
11.1
通讯作者：
Li, Ming
中科院分区：
文献类型：
--
作者：
Huang, Xingyuan;Lu, Ying;Li, Ming
单链DNA断裂酶在断裂DNA的末端沿着沿着DNA（ssDNA）断裂以形成具有类似于18个碱基的周期性的螺旋核丝。丝状体催化同源序列的搜索和检查，并在同源重组（HR）过程中促进与供体双链体的链交换，其机制自发现以来一直是个谜。在这里，通过将错配的片段插入供体双链体并使用单分子技术来捕获HR中的瞬时中间体，我们发现，即使3个碱基对（bp）仍然是基本单位，同源性检查和链交换都可以同时进行多个步骤，导致平均类似于9-bp的大步骤。更有趣的是，链交换被错配片段远程阻断，终止于匹配-错配接头前类似于9 bp的位置。同源性检验和链交换因此在空间上分开，而链交换滞后。我们的数据表明，链交换进展像行波，其中供体DNA被连续纳入ssDNA-RecA丝，以检查同源性，在类似于9-bp的步骤中的前沿，然后由一个假设的过渡段，然后后链交换双链体。
Recombinases polymerize along single-stranded DNA (ssDNA) at the end of a broken DNA to form a helical nucleofilament with a periodicity of similar to 18 bases. The filament catalyzes the search and checking for homologous sequences and promotes strand exchange with a donor duplex during homologous recombination (HR), the mechanism of which has remained mysterious since its discovery. Here, by inserting mismatched segments into donor duplexes and using single-molecule techniques to catch transient intermediates in HR, we found that, even though 3 base pairs (bp) is still the basic unit, both the homology checking and the strand exchange may proceed in multiple steps at a time, resulting in similar to 9-bp large steps on average. More interestingly, the strand exchange is blocked remotely by the mismatched segment, terminating at positions similar to 9 bp before the match-mismatch joint. The homology checking and the strand exchange are thus separated in space, with the strand exchange lagging behind. Our data suggest that the strand exchange progresses like a traveling wave in which the donor DNA is incorporated successively into the ssDNA-RecA filament to check homology in similar to 9-bp steps in the frontier, followed by a hypothetical transitional segment and then the post-strand-exchanged duplex.