The Escherichia coli replisome is inherently DNA damage tolerant

Abstract

The Escherichia coli DNA replication machinery must frequently overcome template lesions under normal growth conditions. Yet, the outcome of a collision between the replisome and a leading-strand template lesion remains poorly understood. Here, we demonstrate that a single, site-specific, cyclobutane pyrimidine dimer leading-strand template lesion provides only a transient block to fork progression in vitro. The replisome remains stably associated with the fork after collision with the lesion. Leading-strand synthesis is then reinitiated downstream of the damage in a reaction that is dependent on the primase, DnaG, but independent of any of the known replication-restart proteins. These observations reveal that the replisome can tolerate leading-strand template lesions without dissociating by synthesizing the leading strand discontinuously.

Fig. 1

A single leading-strand template CPD provides only a transient block to replisome progression. (A) Diagram of the CPD-containing plasmid used as template for replication reactions. (B) Illustration showing the staging of typical bulk (i), and column-isolated (ii), oriC-dependent replication reactions using EcoRI to release topologically-stalled forks. The predicted replication products following post-replication PvuI digestion are shown for the CPD and undamaged templates. (C and D) Time courses for replication reactions conducted in bulk (C) or using column-isolated, replisome-associated ERIs (D). Reaction products were digested with PvuI and analyzed by both native and denaturing gel electrophoresis as indicated. (D) The * denotes products formed from ERIs where the leading strand was labeled but not extended following EcoRI cleavage.

Fig. 2

Restart products are associated with the production of full-length duplex DNA. (A and B) Two-dimensional gel electrophoretic analysis of the replication products from both bulk (A), and column-isolated (B), EcoRI-dependent reactions using the CPD template. Reactions were incubated for 4 min prior to quenching and PvuI cleavage. Samples of the same reactions were run solely through native and alkaline gels to serve as markers for the major reaction products. The * is as in Fig. 1D.

Fig. 3

Restart products are generated by leading-strand synthesis. (A) Inhibition of replication by the Tus/ter complex generates a 50–100 base region of ssDNA on the lagging-strand template (12). Post-replication EcoRI cleavage will release leading-strand products as full-length duplex DNA because the restriction site is located 32 bp to the 5′ side of terB. (B and C) Replication reactions were conducted in bulk on undamaged (B) and CPD (C) templates in the presence of DNA gyrase. Following 6-min incubations, products were digested with EcoRI and PvuI prior to analysis by two-dimensional gel electrophoresis, with additional samples run solely through either native or alkaline gels serving as markers. Uncut refers to the positions of products (stalled forks and products of uncoupled replication) that are not released by EcoRI / PvuI cleavage.

Fig. 4

DnaG re-primes the leading-strand template downstream of the CPD. Column-isolated CPD-template replication reactions were initiated by EcoRI cleavage in the presence or absence of DnaG for 6 min. Reactions were quenched and digested with either PvuI, or PvuI and EagI, which maps to the region of the template downstream of the CPD (fig. S5B). Samples were analyzed by both native and denaturing gel electrophoresis. The * is as in Fig. 1D.