Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA
Termination of DNA replication at Tus-ter barriers results in under-replication of template DNA
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DOI：
10.1016/j.jbc.2021.101409
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发表时间：
2021-12-01
影响因子：
4.8
通讯作者：
Hawkins, Michelle
中科院分区：
文献类型：
--
作者：
Jameson, Katie H.;Rudolph, Christian J.;Hawkins, Michelle
基因组信息的完整和准确复制对于维持所有生命领域的基因组稳定至关重要。在大肠杆菌中，复制终止，即复制过程的最后阶段，被Tus蛋白与基因组位点结合形成的多个单向叉屏障限制在“复制叉陷阱”区域。终止通常发生在远离Tus-ter复合体的地方，但当一个复制体的延迟允许第二个复制体在染色体周围移动超过一半时，它们就成为叉融合过程的一部分。在这种情况下，复制体的进展在Tus-ter复合体的非允许界面被阻断，然后在收敛的复制体遇到允许界面时发生终止。为了研究Tus-ter复合体复制叉融合的后果，我们建立了一个基于质粒的复制系统，我们可以在体外模拟Tus-ter复合体的终止过程。我们开发了一种末端定位试验来测量前导链复制叉的进展，并证明当复制叉在Tus-ter复合体上融合时，DNA模板的复制不足15至24个碱基。这个缺口不能通过添加后链加工酶或包含几种促进DNA复制的解旋酶来弥补。我们的研究结果表明，在Tus-ter屏障上准确的叉融合需要进一步的酶处理，这突出了我们对染色体复制的最后阶段和具有复制叉陷阱的进化优势的理解仍然存在很大的差距。
The complete and accurate duplication of genomic information is vital to maintain genome stability in all domains of life. In Escherichia coli, replication termination, the final stage of the duplication process, is confined to the "replication fork trap" region by multiple unidirectional fork barriers formed by the binding of Tus protein to genomic ter sites. Termination typically occurs away from Tus-ter complexes, but they become part of the fork fusion process when a delay to one replisome allows the second replisome to travel more than halfway around the chromosome. In this instance, replisome progression is blocked at the nonpermissive interface of the Tus-ter complex, termination then occurs when a converging replisome meets the permissive interface. To investigate the consequences of replication fork fusion at Tus-ter complexes, we established a plasmid-based replication system where we could mimic the termination process at Tus-ter complexes in vitro. We developed a termination mapping assay to measure leading strand replication fork progression and demonstrate that the DNA template is under-replicated by 15 to 24 bases when replication forks fuse at Tus-ter complexes. This gap could not be closed by the addition of lagging strand processing enzymes or by the inclusion of several helicases that promote DNA replication. Our results indicate that accurate fork fusion at Tus-ter barriers requires further enzymatic processing, highlighting large gaps that still exist in our understanding of the final stages of chromosome duplication and the evolutionary advantage of having a replication fork trap.