Abstract

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.

Highlights

  • Faithful and efficient DNA replication is a fundamental life process that is the result of complex interactions between a diverse collection of enzymes

  • To better understand the mechanism and cellular impact of helicase regulation, we used CRISPR-Cas9 genome editing to study four previously identified steric exclusion and wrapping (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

  • The constricted conformation is more efficient for translocation and unwinding but is unable to transverse over duplex DNA

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Summary

Introduction

Faithful and efficient DNA replication is a fundamental life process that is the result of complex interactions between a diverse collection of enzymes. Proximal to this process is the DNA helicase enzyme, a hexameric protein ensemble that separates double-stranded DNA (dsDNA) for synthesis and coordinates replicative actions. The excluded strand has been implicated in helicase regulation [4,5,6,7,8,9] by interacting electrostatically on the exterior of many different helicases from multiple organisms to control unwinding speed, establishing the steric exclusion and wrapping (SEW) model for unwinding [10,11,12,13]. Other mechanisms for helicase regulation have been proposed to modulate both the speed of unwinding, coupled unwinding and synthesis, and coordinated priming [14,15,16,17,18,19], but little has been done to examine the cellular consequences of helicase dysregulation in vivo

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