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

The holD gene codes for the yr subunit of the Escherichia coli DNA polymera se III holoenzyme, a component of the gamma complex clamp loader, A holD mu tant was isolated for the first time in a screen for mutations that increas e the frequency of tandem repeat deletions. in contrast to tandem repeat de letions in wild-type strains, deletion events stimulated by the holD mutati on 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 combinati on 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 p resence of RecBCD. We propose that impairment of replication due to the hol D mutation causes the arrest of the entire replisome; consequently, Hollida y junctions are formed by replication fork reversal, and unequal crossing o ver during RecA- and RecBCD-mediated re-incorporation of reversed forks cau ses the hyper-recombination phenotype.