A single-molecule approach to DNA replication in Escherichia coli cells demonstrated that DNA polymerase III is a major determinant of fork speed

Mol Microbiol. 2013 Nov;90(3):584-96. doi: 10.1111/mmi.12386. Epub 2013 Sep 18.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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


  • DNA, Bacterial
  • Escherichia coli Proteins
  • DNA Polymerase III
  • Bromodeoxyuridine
  • Thymidine