The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.
The Consequences of Replicating in the Wrong Orientation: Bacterial Chromosome Duplication without an Active Replication Origin.
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DOI：
10.1128/mbio.01294-15
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发表时间：
2015-11-03
期刊：
影响因子：
6.4
通讯作者：
Rudolph CJ
中科院分区：
文献类型：
--
作者：
Dimude JU;Stockum A;Midgley-Smith SL;Upton AL;Foster HA;Khan A;Saunders NJ;Retkute R;Rudolph CJ
在所有生物体中，染色体复制在特定起点的复制机制的组装阶段受到调节。在大肠杆菌中，DnaA起始蛋白调节oriC处复制叉的组装。这种调节可以被核酸代谢的缺陷破坏。在缺乏RNase HI的细胞中，复制独立于DnaA和oriC启动，可能是在持久的R环。在缺乏RecG的细胞中，假设来源非依赖性合成的机制类似。然而，最近我们提出，这种合成起始于复制叉融合产生的中间体。在这里，我们提出的数据表明，在缺乏RecG或RNase HI的细胞中，起源独立的合成产生不同的机制，表明这两种蛋白在体内具有不同的作用。我们的数据支持RNase HI处理R环的想法，而RecG需要处理复制叉融合中间体。然而，无论如何发起与原点无关的合成，一部分分叉将以与正常方向相反的方向进行。我们表明，由此产生的迎面遇到转录威胁细胞的生存能力，特别是如果发生在高度转录的地区。因此，尽管RecG和RNase HI的功能不同，但它们都是维持复制控制和定向的重要因素。它们的缺失导致严重的复制问题，突出了正常染色体排列的优势，其利用单个起点来控制叉的数量及其相对于转录的方向，以及限定的终止区域来包含叉融合。这种排列的任何改变都会危及细胞周期控制、染色体动力学，并最终危及细胞活力。重要性细胞分裂需要解旋数百万个DNA碱基对，以生成RNA转录物和染色体复制的模板。由于两个流程使用相同的模板，因此不可避免地会发生频繁冲突。为了尽量减少这些冲突的影响，细菌中的转录和复制遵循相同的方向，从而避免正面碰撞。这种同向性是通过对复制开始位置的严格规定来维持的。我们已经使用大肠杆菌作为模型，以调查细胞中的复制起始的定义的位置受到损害。在缺乏RNase HI或RecG的细胞中，复制从定义的复制起点开始，我们讨论了这种合成产生的不同机制。此外，由此产生的叉在与正常相反的方向上进行，从而诱导转录和复制之间的正面碰撞，我们表明，由此产生的后果是严重到足以威胁细胞的生存能力。细胞分裂需要解旋数百万个DNA碱基对，以生成RNA转录本和染色体复制的模板。由于两个流程使用相同的模板，因此不可避免地会发生频繁冲突。为了尽量减少这些冲突的影响，细菌中的转录和复制遵循相同的方向，从而避免正面碰撞。这种同向性是通过严格控制复制的起始位置来维持的。我们已经使用大肠杆菌作为模型，以调查细胞中的复制起始的定义的位置受到损害。在缺乏RNase HI或RecG的细胞中，复制从定义的复制起点开始，我们讨论了这种合成产生的不同机制。此外，由此产生的叉在与正常相反的方向上进行，从而诱导转录和复制之间的正面碰撞，我们表明，由此产生的后果是严重到足以威胁细胞的生存能力。
Chromosome replication is regulated in all organisms at the assembly stage of the replication machinery at specific origins. In Escherichia coli, the DnaA initiator protein regulates the assembly of replication forks at oriC. This regulation can be undermined by defects in nucleic acid metabolism. In cells lacking RNase HI, replication initiates independently of DnaA and oriC, presumably at persisting R-loops. A similar mechanism was assumed for origin-independent synthesis in cells lacking RecG. However, recently we suggested that this synthesis initiates at intermediates resulting from replication fork fusions. Here we present data suggesting that in cells lacking RecG or RNase HI, origin-independent synthesis arises by different mechanisms, indicative of these two proteins having different roles in vivo. Our data support the idea that RNase HI processes R-loops, while RecG is required to process replication fork fusion intermediates. However, regardless of how origin-independent synthesis is initiated, a fraction of forks will proceed in an orientation opposite to normal. We show that the resulting head-on encounters with transcription threaten cell viability, especially if taking place in highly transcribed areas. Thus, despite their different functions, RecG and RNase HI are both important factors for maintaining replication control and orientation. Their absence causes severe replication problems, highlighting the advantages of the normal chromosome arrangement, which exploits a single origin to control the number of forks and their orientation relative to transcription, and a defined termination area to contain fork fusions. Any changes to this arrangement endanger cell cycle control, chromosome dynamics, and, ultimately, cell viability. Importance Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells. Cell division requires unwinding of millions of DNA base pairs to generate the template for RNA transcripts as well as chromosome replication. As both processes use the same template, frequent clashes are unavoidable. To minimize the impact of these clashes, transcription and replication in bacteria follow the same directionality, thereby avoiding head-on collisions. This codirectionality is maintained by a strict regulation of where replication is started. We have used Escherichia coli as a model to investigate cells in which the defined location of replication initiation is compromised. In cells lacking either RNase HI or RecG, replication initiates away from the defined replication origin, and we discuss the different mechanisms by which this synthesis arises. In addition, the resulting forks proceed in a direction opposite to normal, thereby inducing head-on collisions between transcription and replication, and we show that the resulting consequences are severe enough to threaten the viability of cells.