title: Replication fork progression is paused in two large chromosomal zones flanking the DNA replication origin in Escherichia coli.
abstract: 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.
authors.affiliation: Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
authors.forename: Masahiro Tatsumi
authors.initials: MT
authors.lastname: Akiyama
authors.affiliation: Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
authors.forename: Taku
authors.initials: T
authors.lastname: Oshima
authors.affiliation: Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
authors.forename: Onuma
authors.initials: O
authors.lastname: Chumsakul
authors.affiliation: Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
authors.forename: Shu
authors.initials: S
authors.lastname: Ishikawa
authors.affiliation: Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan.
authors.forename: Hisaji
authors.initials: H
authors.lastname: Maki
mesh_terms: D001973:Bromodeoxyuridine; D002876:Chromosomes, Bacterial / Q000235:genetics; D004261:DNA Replication / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D018741:Replication Origin / Q000235:genetics*; D013936:Thymidine / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D001973:Bromodeoxyuridine; D013936:Thymidine