Million-Weaver Samuel, Samadpour Ariana Nakta, Merrikh Houra
Department of Microbiology, University of Washington, Seattle, Washington, USA.
Department of Microbiology, University of Washington, Seattle, Washington, USA Department of Genome Sciences, University of Washington, Seattle, Washington, USA
J Bacteriol. 2015 Jul;197(14):2374-82. doi: 10.1128/JB.00237-15. Epub 2015 May 4.
Efficient duplication of genomes depends on reactivation of replication forks outside the origin. Replication restart can be facilitated by recombination proteins, especially if single- or double-strand breaks form in the DNA. Each type of DNA break is processed by a distinct pathway, though both depend on the RecA protein. One common obstacle that can stall forks, potentially leading to breaks in the DNA, is transcription. Though replication stalling by transcription is prevalent, the nature of DNA breaks and the prerequisites for replication restart in response to these encounters remain unknown. Here, we used an engineered site-specific replication-transcription conflict to identify and dissect the pathways required for the resolution and restart of replication forks stalled by transcription in Bacillus subtilis. We found that RecA, its loader proteins RecO and AddAB, and the Holliday junction resolvase RecU are required for efficient survival and replication restart after conflicts with transcription. Genetic analyses showed that RecO and AddAB act in parallel to facilitate RecA loading at the site of the conflict but that they can each partially compensate for the other's absence. Finally, we found that RecA and either RecO or AddAB are required for the replication restart and helicase loader protein, DnaD, to associate with the engineered conflict region. These results suggest that conflicts can lead to both single-strand gaps and double-strand breaks in the DNA and that RecA loading and Holliday junction resolution are required for replication restart at regions of replication-transcription conflicts.
Head-on conflicts between replication and transcription occur when a gene is expressed from the lagging strand. These encounters stall the replisome and potentially break the DNA. We investigated the necessary mechanisms for Bacillus subtilis cells to overcome a site-specific engineered conflict with transcription of a protein-coding gene. We found that the recombination proteins RecO and AddAB both load RecA onto the DNA in response to the head-on conflict. Additionally, RecA loading by one of the two pathways was required for both replication restart and efficient survival of the collision. Our findings suggest that both single-strand gaps and double-strand DNA breaks occur at head-on conflict regions and demonstrate a requirement for recombination to restart replication after collisions with transcription.
基因组的有效复制依赖于起始点以外复制叉的重新激活。重组蛋白可促进复制重新启动，尤其是当DNA中形成单链或双链断裂时。每种类型的DNA断裂都通过不同的途径进行处理，不过两者都依赖于RecA蛋白。一个可能使复制叉停滞并可能导致DNA断裂的常见障碍是转录。尽管转录导致的复制停滞很普遍，但DNA断裂的性质以及响应这些情况进行复制重新启动的先决条件仍不清楚。在这里，我们利用一个工程化的位点特异性复制-转录冲突，来识别和剖析枯草芽孢杆菌中因转录而停滞的复制叉的解决和重新启动所需的途径。我们发现，在与转录发生冲突后，高效存活和复制重新启动需要RecA、其加载蛋白RecO和AddAB以及霍利迪连接体解离酶RecU。遗传分析表明，RecO和AddAB并行作用，以促进RecA在冲突位点的加载，但它们各自可以部分弥补对方的缺失。最后，我们发现复制重新启动和螺旋酶加载蛋白DnaD与工程化冲突区域结合需要RecA和RecO或AddAB中的一种。这些结果表明，冲突可导致DNA中的单链缺口和双链断裂，并且在复制-转录冲突区域进行复制重新启动需要RecA加载和霍利迪连接体解离。
当一个基因从滞后链表达时，复制和转录之间会发生迎头冲突。这些情况会使复制体停滞并可能使DNA断裂。我们研究了枯草芽孢杆菌细胞克服与一个蛋白质编码基因转录的位点特异性工程冲突所需的必要机制。我们发现，重组蛋白RecO和AddAB都会因迎头冲突而将RecA加载到DNA上。此外，两种途径之一的RecA加载对于复制重新启动和碰撞后的高效存活都是必需的。我们的研究结果表明，单链缺口和双链DNA断裂都发生在迎头冲突区域，并证明了在与转录碰撞后重新启动复制需要重组。
