Repair of DNA damage begins with the elicitation of targeted cellular responses to restore the genome. In E. coli, major products of DNA damage result in the buildup of single-stranded DNA (ssDNA) that is rapidly bound by cooperative filamentation of RecA to initiate the SOS response. The replicative helicase, DnaB, is a central component of the replisome, unwinding duplex DNA in concert with Pol III template dependent synthesis. Interestingly, helicase unwinding is heavily regulated, and the unwinding rate can be reduced by over 10-fold if DnaB becomes decoupled from Pol III. However, if DnaB is dysregulated by mutations that enforce a faster more constricted conformation, unwinding can continue independently, generating excess ssDNA resulting in severe cellular stress. This surplus ssDNA can stimulate RecA recruitment for recombinational repair or activation of SOS to increase the available repair protein pool. To better understand the consequences of dysregulated unwinding, we combined targeted dnaB mutations with an inducible plasmid-based RecA filament inhibition strategy to examine the dependencies on RecA in counteracting decoupling. We find that RecA filamentation is instrumental for processing daughter strand gaps left behind from decoupled unwinding and synthesis to prevent DNA breaks. Without functional RecA filaments, dnaB mutant strains had a greater burden from endogenous damage but without a compensatory increase in mutagenesis. Overall, RecA plays a critical role in strain survival by processing DNA gaps and protecting from breaks caused by dysregulated or interrupted helicase activity in vivo.

DNA replication is a vital process ensuring the passing down of intact genetic information over generations. DNA replication in the model organism, Escherichia coli (E. coli), has been extensively studied, providing significant insights into the various enzyme mechanisms. However, while the prime functions of individual components of the replisome have been deciphered, the more complex interactions between the components is an active area of study to understand replisome coupling. At the core of the replisome, the DnaB helicase unwinds the double strand DNA which is followed by rapid synthesis of nascent strand by Polymerase III core (Pol III core). It is proposed that the Tau subunit of the clamp loader complex acts as the physical link between both DnaB and Pol III core, leading to a coupling phenomenon between unwinding and synthesis. Using a reconstituted E. coli replisome in vitro, unwinding of DNA substrates with variable lengths and assemblies can be monitored through electrophoresis to better understand the kinetics of coupling. Mutations have been introduced in the exterior surface of DnaB, disrupting its charged interactions with the excluded strand, and promoting more constricted conformation that result in faster unwinding. The unwinding kinetics of wild‐type versus mutant DnaB enzymes on different length substrates are being examined. Further experiments include Pol III synthesis alone or with proposed coupling from the tau‐subunit and can simultaneously examine unwinding and synthesis. Mutations that affect DnaB conformation or disrupt Pol III‐tau interactions will be utilized to assess their relative influences in maintaining a coupled replisome. Conducting in vitro experiments will provide mechanistic insight into the nature of interactions between these replisome components in a coordinated system. Coupling interactions are also being investigated in vivo using precise CRISPR‐Cas9 genome editing. Coupling mutations have been introduced in the dnaX gene encoding the tau‐subunit. These mutant strains exhibit a reduced growth rate when compared to wild‐type strains, indicating that disrupted coupling leads to reduced doubling times. These strains are being further characterized to determine the genomic and cellular consequences of helicase‐polymerase decoupling in vivo. Therefore, a combination of in vitro and in vivotechniques are being used to better understand the importance of coupling during DNA replication to maintain a stable genome.