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

Author:

McKenzie Aidan M.,Henry CamilleORCID,Myers Kevin S.,Place Michael M.,Keck James L.ORCID

Abstract

AbstractCollisions 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 ΔpriB 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.Author SummaryAll organisms rely on DNA replication to grow, develop, and reproduce. In bacteria, the cellular machinery that carries out DNA replication is estimated to fail and prematurely dissociate from the genome at least once per cell cycle. As a result, bacteria have evolved “DNA replication restart” mechanisms that resuscitate failed replication reactions. To probe the function and context of DNA replication restart in the bacterium Escherichia coli, we employed a genetic screen to identify genes that were conditionally important in mutant E. coli strains compromised in their ability to perform DNA replication restart. Identification of genes with previously known relationships with DNA replication restart confirmed the robustness of our screen, while additional findings implicated novel genetic relationships. Targeted experiments validated the importance of these genes and provided an explanation for their significance in preventing double-strand DNA breaks in cells, a severe form of DNA damage. Our results help to define specific roles for the genes identified by our screen and elucidate the contextual environment of DNA repair upstream of DNA replication restart in E. coli.

Publisher

Cold Spring Harbor Laboratory

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