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首页> 外文期刊>Genes to cells : >Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli
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Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli

机译：复制叉的进程在大肠杆菌DNA复制起点两侧的两个大染色体区域中被暂停

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摘要

Although the speed of nascent DNA synthesis at individual replication forks is relatively uniform in bacterial cells, the dynamics of replication fork progression on the chromosome are hampered by a variety of natural impediments. Genome replication dynamics can be directly measured from an exponentially growing cell population by sequencing newly synthesized DNA strands that were specifically pulse-labeled with the thymidine analogue 5-bromo-2-deoxyuridine (BrdU). However, a short pulse labeling with BrdU is impracticable for bacteria because of poor incorporation of BrdU into the cells, and thus, the genomewide dynamics of bacterial DNA replication remain undetermined. Using a new thymidine-requiring Escherichia coli strain, eCOMB, and high-throughput sequencing, we succeeded in determining the genomewide replication profile in bacterial cells. We also found that fork progression is paused in two similar to 200-kb chromosomal zones that flank the replication origin in the growing cells. This origin-proximal obstruction to fork progression was overcome by an increased thymidine concentration in the culture medium and enhanced by inhibition of transcription. These indicate that DNA replication near the origin is sensitive to the impediments to fork progression, namely a scarcity of the DNA precursor deoxythymidine triphosphate and probable conflicts between replication and transcription machineries.
机译：尽管细菌细胞中单个复制叉处新生DNA合成的速度相对均匀，但是染色体上复制叉进展的动力学受到多种自然障碍的阻碍。通过对新合成的DNA链进行测序，可以直接从指数增长的细胞群体中测量基因组复制动态，这些DNA链特别用胸苷类似物5-溴-2-脱氧尿苷（BrdU）进行了脉冲标记。但是，由于BrdU掺入细胞的能力较差，因此用BrdU进行短脉冲标记对于细菌是不可行的，因此细菌DNA复制的全基因组动力学仍然不确定。使用新的需要胸腺嘧啶核苷的大肠杆菌菌株eCOMB和高通量测序，我们成功地确定了细菌细胞中的全基因组复制谱。我们还发现，叉子生长在两个相似的200kb染色体区域（位于生长细胞中的复制起点侧翼）处被暂停。通过增加培养基中的胸苷浓度和通过抑制转录而增强，可以克服这种起源于叉叉的近端梗阻。这些表明在原点附近的DNA复制对叉进展的障碍敏感，即DNA前体脱氧胸苷三磷酸的缺乏以及复制和转录机制之间可能的冲突。

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