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

Abstract

SummaryThe 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 ). This indicates that the speed of chain elongation by Pol III is a major determinant of replication fork speed in E. coli cells.

Template-switching during replication fork repair in bacteria

“…In the rpsL gene, some QPM sites are strongly induced by a mutation affecting the DNA Pol III polymerase subunit ( dnaE173 ) of DNA pol III [102]. The dnaE173 mutant exhibits slow replication elongation rates in vivo [116] and the purified core polymerase exhibits a low Km for nucleotide and a proofreading deficit [117]. Mutagenesis at thyA QPM hotspot is elevated 11-fold by the loss of all three of E. coli ‘ s DNA damage induced polymerases, Pol II, Pol IV and PolV; no single or double mutant shows an effect, suggesting that all three polymerases function in a redundant fashion for QPM avoidance [112].…”

The Precarious Prokaryotic Chromosome

“…The in vitro rate of purified main bacterial replicase DNA polymerase (pol) III is ϳ500 nt/s (155)(156)(157). The directly measured rates of replication fork propagation in vivo, at 620 to 700 nt/s at 30°C (158, 159) and 1,300 nt/s at 42°C (159), are even higher, and there is evidence that the rate is limited by the rate of DNA pol III chain elongation (160). In contrast, the maximal rate of the yeast leading-strand DNA polymerase epsilon in vitro is only 50 nt/s, although under the same conditions the lagging-strand DNA polymerase delta moves faster, at 200 nt/s (157).…”

Independent and Stochastic Action of DNA Polymerases in the Replisome

“…The Escherichia coli genome is replicated at ~650 bp•s −1 in vivo (Pham et al, 2013) by a replisome comprising at least thirteen distinct polypeptides. The hexameric helicase DnaB, which translocates 5′→3′ on the lagging-strand template, unwinds DNA at the replication fork.…”