Targeted chromosomal Escherichia coli:dnaB exterior surface residues regulate DNA helicase behavior to maintain genomic stability and organismal fitness.

Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression. Currently, mechanisms for helicase regulation that involve interactions with both DNA strands through a steric exclusion and wrapping (SEW) model and conformational shifts between dilated and constricted states have been examined in vitro. To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified SEW-deficient mutants of the bacterial replicative helicase DnaB. We discovered that these four SEW mutations stabilize constricted states, with more fully constricted mutants having a generally greater impact on genomic stress, suggesting a dynamic model for helicase regulation that involves both excluded strand interactions and conformational states. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. Specifically, dnaB:mut strains present with increased mutational frequencies without significantly inducing SOS, consistent with leaving single-strand gaps in the genome during replication that are subsequently filled with lower fidelity. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving a spectrum of DnaB conformational changes and relates current mechanistic understanding to functional helicase behavior at the replication fork.

Contacts and context that regulate DNA helicase unwinding and replisome progression.

Hexameric DNA helicases involved in the separation of duplex DNA at the replication fork have a universal architecture but have evolved from two separate protein families. The consequences are that the regulation, translocation polarity, strand specificity, and architectural orientation varies between phage/bacteria to that of archaea/eukaryotes. Once assembled and activated for single strand DNA translocation and unwinding, the DNA polymerase couples tightly to the helicase forming a robust replisome complex. However, this helicase-polymerase interaction can be challenged by various forms of endogenous or exogenous agents that can stall the entire replisome or decouple DNA unwinding from synthesis. The consequences of decoupling can be severe, leading to a build-up of ssDNA requiring various pathways for replication fork restart. All told, the hexameric helicase sits prominently at the front of the replisome constantly responding to a variety of obstacles that require transient unwinding/reannealing, traversal of more stable blocks, and alternations in DNA unwinding speed that regulate replisome progression.