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- Title
A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed.
- Authors
Pham, Tuan Minh; Tan, Kang Wei; Sakumura, Yuichi; Okumura, Katsuzumi; Maki, Hisaji; Akiyama, Masahiro Tatsumi
- 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.
- Subjects
DNA replication; ESCHERICHIA coli; DNA polymerases; THYMIDINE; SCANNING electron microscopy; BACTERIAL cells
- Publication
Molecular Microbiology, 2013, Vol 90, Issue 3, p584
- ISSN
0950-382X
- Publication type
Article
- DOI
10.1111/mmi.12386