title: A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed.
abstract: The replisome catalyses DNA synthesis at a DNA replication fork. The molecular behaviour of the individual replisomes, and therefore the dynamics of replication fork movements, in growing Escherichia coli cells remains unknown. DNA combing enables a single-molecule approach to measuring the speed of replication fork progression in cells pulse-labelled with thymidine analogues. We constructed a new thymidine-requiring strain, eCOMB (E. coli for combing), that rapidly and sufficiently incorporates the analogues into newly synthesized DNA chains for the DNA-combing method. In combing experiments with eCOMB, we found the speed of most replication forks in the cells to be within the narrow range of 550-750 nt s(-1) and the average speed to be 653 ± 9 nt s(-1) (± SEM). We also found the average speed of the replication fork to be only 264 ± 9 nt s(-1) in a dnaE173-eCOMB strain producing a mutant-type of the replicative DNA polymerase III (Pol III) with a chain elongation rate (300 nt s(-1) ) much lower than that of the wild-type Pol III (900 nt s(-1) ). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.
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: Tuan Minh
authors.initials: TM
authors.lastname: Pham
authors.forename: Kang Wei
authors.initials: KW
authors.lastname: Tan
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Sakumura
authors.forename: Katsuzumi
authors.initials: K
authors.lastname: Okumura
authors.forename: Hisaji
authors.initials: H
authors.lastname: Maki
authors.forename: Masahiro Tatsumi
authors.initials: MT
authors.lastname: Akiyama
mesh_terms: D001973:Bromodeoxyuridine; D002876:Chromosomes, Bacterial; D004258:DNA Polymerase III / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D013936:Thymidine / Q000031:analogs & derivatives
chemical_list: D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D004258:DNA Polymerase III; D001973:Bromodeoxyuridine; D013936:Thymidine