A single‐molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed |
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| Authors: | Tuan Minh Pham Kang Wei Tan Yuichi Sakumura Katsuzumi Okumura Hisaji Maki Masahiro Tatsumi Akiyama |
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| Institution: | 1. Division of Systems Biology, Graduate School of Biological Sciences, Nara Institute of Science and Technology, , Ikoma, Nara, 630‐0192 Japan;2. Department of Information Science and Technology, Aichi Prefectural University, , Nagakute, Aichi, 480‐1198 Japan;3. Department of Life Science, Graduate School of Bioresources, Mie University, , Tsu, Mie, 514‐8507 Japan |
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| 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. |
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