Identification of genetic interactions with priB links the PriA/PriB DNA replication restart pathway to double-strand DNA break repair in Escherichia coli

Abstract

Collisions between DNA replication complexes (replisomes) and impediments such as damaged DNA or proteins tightly bound to the chromosome lead to premature dissociation of replisomes at least once per cell cycle in Escherichia coli. Left unrepaired, these events produce incompletely replicated chromosomes that cannot be properly partitioned into daughter cells. DNA replication restart, the process that reloads replisomes at prematurely terminated sites, is therefore essential in E. coli and other bacteria. Three replication restart pathways have been identified in E. coli: PriA/PriB, PriA/PriC, and PriC/Rep. A limited number of genetic interactions between replication restart and other genome maintenance pathways have been defined, but a systematic study placing replication restart reactions in a broader cellular context has not been performed. We have utilized transposon insertion sequencing to identify new genetic interactions between DNA replication restart pathways and other cellular systems. Known genetic interactors with the priB replication restart gene (uniquely involved in the PriA/PriB pathway) were confirmed and several novel priB interactions were discovered. Far fewer connections were found with the PriA/PriC or PriC/Rep pathways, suggesting a primacy role for the PriA/PriB pathway in E. coli. Targeted genetic and imaging-based experiments with priB and its genetic partners revealed significant double-strand DNA break (DSB) accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in Δ priB cells. Taken together, our results highlight roles for several genes in DSB homeostasis and define a genetic network that facilitates DNA repair/processing upstream of PriA/PriB-mediated DNA replication restart in E. coli.