Impairment of lagging strand synthesis triggers the formation of a RuvABC substrate at replication forks

Abstract

The holD gene codes for the psi subunit of the Escherichia coli DNA polymerase III holoenzyme, a component of the gamma complex clamp loader. A holD mutant was isolated for the first time in a screen for mutations that increase the frequency of tandem repeat deletions. In contrast to tandem repeat deletions in wild-type strains, deletion events stimulated by the holD mutation require RecA. They do not require RecF, and hence do not result from the recombinational repair of gaps, arguing against uncoupling of the leading and lagging strand polymerases in the holD mutant. The holD recBC combination of mutations is lethal and holD recBts recCts strains suffer DNA double-strand breaks (DSBs) at restrictive temperature. DSBs require the presence of the Holliday junction-specific enzymes RuvABC and are prevented in the presence of RecBCD. We propose that impairment of replication due to the holD mutation causes the arrest of the entire replisome; consequently, Holliday junctions are formed by replication fork reversal, and unequal crossing over during RecA- and RecBCD-mediated re-incorporation of reversed forks causes the hyper-recombination phenotype.

Fig. 1. UV sensitivity of recF mutants is not significantly affected by the holD mutation. Exponential cultures of cells grown to OD 0.5 were plated at appropriate dilutions on minimal medium plates that were irradiated at 2 J/m² for various times and incubated for 48 h at 37°C. Ratios of c.f.u. (colony-forming units) on irradiated versus non-irradiated plates are shown. Open circles, JJC520 (wild type); closed circles, JJC561 (recF400::Tn5); open squares, JJC947 (holD); closed squares, JJC1164 (holD recF400::Tn5). Results are the average of 3–6 independent determinations.

Fig. 2. In recA strains, extensive DNA degradation is expected if blocked replication forks are directly broken, not if they form a Holliday junction. (A) The fork is directly broken. (B) A Holliday junction is formed.

Fig. 3. DNA degradation in recA strains is not significantly affected by the holD mutation. DNA degradation was measured at different times as described previously (see Materials and methods; Seigneur et al., 1998). Open circles, JJC520 (wild type); closed circles, JJC557 (recA); open squares, JJC867 (holD); closed squares, JJC868 (holD recA). Results are the average of 3–5 independent determinations.

Fig. 4. Model for processing of arrested replication forks by recombination proteins in the holD mutants (adapted from Seigneur et al., 1998). In step A, the replication fork is blocked due to a defect in the lagging strand polymerase in the holD mutant. The two newly synthesized strands anneal, forming a Holliday junction that is stabilized by RuvAB binding (step B). Pathway C: RecBCD binds to the double-stranded tail (C1) and initiates a genetic exchange mediated by RecA (C2), RuvC resolves the first Holliday junction bound by RuvAB (C3). Pathway D: RecBCD-mediated degradation of the tail progresses up to the RuvAB-bound Holliday junction. Replication can restart when RecBCD has displaced the RuvAB complex. Pathway E: RuvC resolves the RuvAB-bound Holliday junction. Breakage at both forks results in the linear DNA detected by PFGE. Continuous and discontinuous lines represent the template and the newly synthesized strands of the chromosome, respectively; the arrow indicates the 3′ end of the growing strand.