Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli
Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli
复制标题
大肠杆菌中 DNA 复制起点两侧的两个大染色体区域中的复制叉进程暂停
DOI：
10.1111/gtc.12388
复制
发表时间：
2016
期刊：
影响因子：
2.1
通讯作者：
Shu Ishikawa and Hisaji Mak
中科院分区：
文献类型：
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作者：
Masahiro Tatsumi Akiyama;Taku Oshima;Onuma Chumsakul;Shu Ishikawa and Hisaji Mak
虽然在细菌细胞中，单个复制叉处新生DNA合成的速度相对均匀，但染色体上复制叉进展的动力学受到各种天然障碍的阻碍。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中测量基因组复制动力学，所述DNA链用胸苷类似物5-溴-2 ′-脱氧尿苷（BrdU）进行特异性脉冲标记。然而，短脉冲标记BrdU是不切实际的细菌，因为穷人的BrdU纳入细胞，因此，细菌DNA复制的全基因组动力学仍未确定。使用一种新的胸苷需要大肠杆菌菌株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，分叉进程在生长细胞中复制起点两侧的两个~200-kb染色体区域中暂停。通过增加培养基中的胸苷浓度克服了对分叉进展的起源近端阻碍，并通过抑制转录增强。这些表明，在原点附近的DNA复制是敏感的叉进展的障碍，即缺乏的DNA前体脱氧胸苷三磷酸和复制和转录机制之间可能的冲突。
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‐requiringEscherichia colistrain, 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 ~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.