Dense populations of cells are indicated with red, moderate with green, and minor or diffuse population with blue. FITC indicates the chromatin staining intensity, and FSC indicates cell size. FITC versus FSC (forward scatter) plot of cell cultures exposed to rifampicin and cephalexin analyzed by FACS. S6 Fig: Rifampicin ‘run-out’ FACS reveals unique concentrations of DNA per cell size for dnaB:mut strains. 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. 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. These dnaB mutations result in increased chromosome complexities, less stable genomes, and ultimately less viable and fit strains. 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. 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. 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. Helicase regulation involves modulation of unwinding speed to maintain coordination of DNA replication fork activities and is vital for replisome progression.
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