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

Abstract

Helicase regulation is vital for replisome progression, where the helicase enzyme functions to unwind duplex DNA and aids in the coordination of replication fork activities. 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 DNA damage and chromosome complexity, less stable genomes, and ultimately less viable and fit strains. Notably, while two mutations stabilized fully constricted states, they have distinct effects on genomic stability, suggesting a complex relationship between helicase regulation mechanisms and faithful, efficient DNA replication. This work explores the genomic impacts of helicase dysregulation in vivo, supporting a combined dynamic regulatory mechanism involving SEW and conformational changes and relates current mechanistic understanding to functional helicase behavior.