RecA Utilization During Dysregulated Unwinding in the Prokaryotic Replisome

DNA replication is an essential process for the inheritance of genetic information between generations. Studying the replisome of Escherichia coli provides insights into DnaB helicase unwinding mechanisms and efficient coupling with DNA synthesis by Pol III. Uncoupling at the replisome results in an accumulation of labile and fragile ssDNA, leading to deletions, mutations, and chromosomal rearrangements. During ssDNA buildup, the RecA protein activates the transcriptional SOS response to restore genomic integrity, but this mechanistic process is not completely understood in relation to uncoupling and dysregulated unwinding. To determine the importance of RecA on mitigating uncoupling defects, we utilized dnaB:K180A and dnaB:R328/329A mutations that enforced a constricted state and dysregulated unwinding in combination with an inducible system that inhibits RecA filamentation on ssDNA. When exposed to exogenous damage agents, the results of minimum inhibitory concentration (MIC) and genotoxin survival assays demonstrated that dnaB mutants utilized RecA activation of the SOS response to mitigate dysregulated unwinding and the consequences of ssDNA accumulation in vivo. Further studies can investigate the prevalence and importance of specific RecA mediated pathways for restart after replisome uncoupling is induced by various intrinsic and extrinsic factors.