A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed. Issue 3 (18th September 2013)

• Record Type: Journal Article
• Authors: Pham, Tuan Minh; Tan, Kang Wei; Sakumura, Yuichi; Okumura, Katsuzumi; Maki, Hisaji; Akiyama, Masahiro Tatsumi
• Is Part Of: Molecular microbiology. Volume 90:Issue 3(2013)
• Page Start: 584
• Page End: 596
• Publication Date: 2013-09-18
• Subjects: Molecular microbiology -- Periodicals; 572.829
• DOI: 10.1111/mmi.12386
• Languages: English
• ISSNs: 0950-382X

Abstract

Summary: 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.