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. Author summary: DNA replication is a vital biological process, and the proteins involved are structurally and functionally conserved across all domains of life. As our fundamental knowledge of genes and genetics grows, so does our awareness of links between acquired genetic mutations and disease. Understanding how genetic material is replicated accurately and efficiently and with high fidelity is the foundation to identifying and solving genome-based diseases. E. coli are model organisms, containing core replisome proteins, but lack the complexity of the human replication system, making them ideal for investigating conserved replisome behaviors. The helicase enzyme acts at the forefront of the replication fork to unwind the DNA helix and has also been shown to help coordinate other replisome functions. In this study, we examined specific mutations in the helicase that have been shown to regulate its conformation and speed of unwinding. We investigate how these mutations impact the growth, fitness, and cellular morphology of bacteria with the goal of understanding how helicase regulation mechanisms affect an organism's ability to survive and maintain a stable genome. [ABSTRACT FROM AUTHOR]

Copyright of PLoS Genetics is the property of Public Library of Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)