Regulation, replication, and roadblocks : mechanistic and functional insights into E. coli helicase regulation and coupling in a dynamic replisome.

DNA replication is a process fundamental to life, and yet much about the mechanisms and proteins involved remain to be fully understood. Proximal to the replication fork is the helicase enzyme, responsible for separating duplex DNA into single strands. Because of high conservation of replicative enzymes across Domains, the bacterial helicase DnaB shares elements of both structural and functional homology to eukaryotic replicative helicases and is critical for coordination of fork activities. Regulation of the helicase is vital for fast, efficient, and faithful genome replication, but yet the mechanism for regulation of unwinding is still under debate. 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. However, to better understand the mechanism in an active system and the cellular impact of helicase regulation, targeted genomic dnaB mutations were made using CRISPR-Cas9 to investigate the mechanism and downstream effects of helicase regulation in living organisms. We present data that reveals a dual regulation mechanism involving conformational changes within the hexamer and excluded strand effects and show that eliminating helicase regulation by these mechanisms leads to high genomic stress, mutations, and ultimately cell death. We investigated the downstream chromosomal architecture and confirm that dysregulated unwinding leads to daughter strand gaps that are prone to DNA damage and stimulate mutagenic repair. The homologous recombination protein, RecA, plays a critical role in processing the gaps and breaks left in the wake of rapid, uncontrolled unwinding at the fork. This work explores the genomic impacts of helicase dysregulation at and downstream of the replication fork, expanding current mechanistic understanding and relating it to functional helicase behavior in vivo.