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
; 90(3): 584-96, 2013 Nov.
Article
em En
| MEDLINE
| ID: mdl-23998701
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
DNA Bacteriano
/
DNA Polimerase III
/
Replicação do DNA
/
Escherichia coli
Idioma:
En
Revista:
Mol Microbiol
Assunto da revista:
BIOLOGIA MOLECULAR
/
MICROBIOLOGIA
Ano de publicação:
2013
Tipo de documento:
Article
País de afiliação:
Japão