SummaryEscherichia coli dinB encodes the specialized DNA polymerase DinB (Pol IV), which is induced as part of the SOS stress-response system and functions in translesion synthesis (TLS) to relieve the replicative Pol III that is stalled at DNA lesions. As the number of DinB molecules, even in unstressed cells, is greater than that required to accomplish TLS, it is thought that dinB plays some additional physiological role. Here, we overexpressed dinB under the tightly regulable arabinose promoter and looked for a distinct phenotype. Upon induction of dinB expression, progression of the replication fork was immediately inhibited at random genomic positions, and the colony-forming ability of the cells was reduced. Overexpression of mutated dinB alleles revealed that the structural requirements for these two inhibitory effects and for TLS were distinct. The extent of in vivo inhibition displayed by a mutant DinB matched the extent of its in vitro impedance, at near-physiological concentration, of a moving Pol III. We suggest that DinB targets Pol III, thereby acting as a brake on replication fork progression. Because the brake operates when cells have excess DinB, as they do under stress conditions, it may serve as a checkpoint that modulates replication to safeguard genome stability.

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DNA polymerases δ δ δ δ and ε ε ε ε (Polδ δ δ δ and Polε ε ε ε ) are widely thought to be the major DNA polymerases that function in elongation during DNA replication in eukaryotic cells. However, the precise roles of these polymerases are still unclear. Here we comparatively analysed DNA replication in Xenopus egg extracts in which Polδ δ δ δ or Polε ε ε ε was immunodepleted. Depletion of either polymerase resulted in a significant decrease in DNA synthesis and accumulation of short nascent DNA products, indicating an elongation defect. Moreover, Polδ δ δ δ depletion caused a more severe defect in elongation, as shown by sustained accumulation of both short nascent DNA products and singlestranded DNA gaps, and also by elevated chromatin binding of replication proteins that function more frequently during lagging strand synthesis. Therefore, our data strongly suggest the possibilities that Polδ δ δ δ is essential for lagging strand synthesis and that this function of Polδ δ δ δ cannot be substituted for by Polε ε ε ε .

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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.