Akiyama Masahiro Tatsumi, Oshima Taku, Chumsakul Onuma, Ishikawa Shu, Maki Hisaji
Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
Genes Cells. 2016 Aug;21(8):907-14. doi: 10.1111/gtc.12388. Epub 2016 Jun 28.
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 ~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.
尽管在细菌细胞中，单个复制叉处新生DNA合成的速度相对均匀，但染色体上复制叉前进的动态过程受到多种自然阻碍的影响。通过对用胸苷类似物5-溴-2'-脱氧尿苷（BrdU）特异性脉冲标记的新合成DNA链进行测序，可以直接从指数生长的细胞群体中测量基因组复制动态。然而，由于BrdU掺入细胞的效率较低，对细菌进行短时间的BrdU脉冲标记是不可行的，因此，细菌DNA复制的全基因组动态仍未确定。我们使用一种新的需要胸苷的大肠杆菌菌株eCOMB和高通量测序技术，成功地确定了细菌细胞中的全基因组复制图谱。我们还发现，在生长细胞中，复制叉的前进在两个位于复制起点两侧约200 kb的染色体区域中暂停。培养基中胸苷浓度的增加克服了这种靠近起点的对复制叉前进的阻碍，而转录抑制则增强了这种阻碍。这些结果表明，起点附近的DNA复制对复制叉前进的阻碍敏感，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存在的冲突。
