PRJNA837116
Overwhelming DNA replication restart in Escherichia coli with double-stranded breaks
DNA replication complexes (replisomes) are challenged by damaged DNA and tightly bound proteins throughout the chromosome duplication process. These encounters prematurely dissociate replisomes at an estimated frequency of at least once per cell cycle in Escherichia coli. Left unrepaired, this situation leaves an incompletely replicated chromosome that will prevent proper cell division. DNA replication restart, the process that reloads the replisome at premature termination sites, is therefore essential in E. coli and other bacteria. Multiple replication restart pathways have been identified in E. coli and the key proteins that mediate the process are known: PriA, PriB, PriC, DnaT, and Rep. A limited number of genetic interactions between replication restart factors and other genome maintenance pathways have been defined, but an unbiased study placing replication restart reactions in a broader cellular context has not been performed. Here we utilized transposon insertion sequencing (Tn-seq) to systematically identify new genetic interactions between DNA replication restart genes and other systems in E. coli. Our genetic screen confirmed known genetic interactors with the replication restart gene, priB, while it also identified many novel interactions with a variety of other genes. Subsequent targeted genetic and microscopy experiments into priB and its genetic partners revealed significant double-strand DNA break accumulation in strains with mutations in dam, rep, rdgC, lexA, or polA. Notably, modulating the activity of the RecA recombinase partially suppressed the detrimental effects of rdgC or lexA mutations in delta-priB cells. These results highlight roles for several genes in double-strand break homeostasis and define the genetic network that is critical for DNA repair/processing upstream of DNA replication restart in E. coli.