A single‐molecule approach to <scp>DNA</scp> replication in <i><scp>E</scp>scherichia coli</i> cells demonstrated that <scp>DNA</scp> polymerase <scp>III</scp> is a major determinant of fork speed

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 E scherichia 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.