| RRC ID |
85521
|
| Author |
Pham TM, Tan KW, Sakumura Y, Okumura K, Maki H, Akiyama MT.
|
| Title |
A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed.
|
| Journal |
Mol Microbiol
|
| 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.
|
| Volume |
90(3)
|
| Pages |
584-96
|
| Published |
2013-11-1
|
| DOI |
10.1111/mmi.12386
|
| PMID |
23998701
|
| MeSH |
Bromodeoxyuridine
Chromosomes, Bacterial
DNA Polymerase III / genetics
DNA Polymerase III / metabolism*
DNA Replication*
DNA, Bacterial / biosynthesis*
DNA, Bacterial / metabolism
Escherichia coli / genetics
Escherichia coli / growth & development*
Escherichia coli / metabolism
Escherichia coli Proteins / genetics
Escherichia coli Proteins / metabolism
Mutation
Thymidine / analogs & derivatives
|
| IF |
3.418
|
| Resource |
| Prokaryotes E. coli |
ME10091 |
