Dysregulated DnaB unwinding induces replisome decoupling and daughter strand gaps that are countered by RecA polymerization

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. AUTHOR SUMMARYCoupled DNA unwinding and synthesis is a genomic protection strategy used during DNA replication to prevent excessive buildup of labile single-stranded DNA (ssDNA). The helicase and polymerase enzymes have evolved multidimensional regulation tactics to maintain this connection despite different individual kinetic rates and differential responses to genomic obstacles. For one, the DnaB helicase in E. coli can alter its hexameric ring structure by dilating to slow down or constricting to speed up DNA unwinding. Here, we have utilized persistently constricted mutants of DnaB in vitro or genomically edited dnaB in vivo to induce decoupling in the replisome. Constricted DnaB mutants limit total leading strand synthesis by Pol III, indicating that lost kinetic regulation between these enzymes results in inefficient replication. Using an inducible plasmid-based system to disrupt Rad51 filamentation in vivo, we show that RecA is responsible for the increased mutagenesis, a filamented cellular phenotype, and mitigating DNA breaks from excess ssDNA caused by decoupling. These results reveal a role for RecA filamentation in mediating excess ssDNA resulting from decoupling to maintain survival and adaptation.