No 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.

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.

The average American consumes 300mg of caffeine a day, and only about 70% is metabolized into Theophylline and Theobromine. The other 30% is excreted primarily through urine, where it goes to water treatment plants and then into the environment. Previous studies have shown that most water treatment plants are capable of removing 90-95% of the caffeine during treatment, but the remainder enters the environment without being broken down.Caffeine is the one of the most widely consumed and least-regulated drugs. Due to the molecule’s integrity, it has potential to build up in water systems over time. Even microgram per liter amounts have been shown to cause significant effects in small aquatic invertebrates and shellfish, and may ultimately affect the ecosystem as a whole. In addition to the environmental affects, if caffeine is present in drinking water, it could have significant impact for those with heart conditions and those with sensitivity to caffeine. In this study, samples were collected from local streams and rivers as well as city and well water taps. They were analyzed using LC/MS/MS to quantify the caffeine, theobromine, and theophylline content using a deuterated internal standard, and a comparative study was performed. The observed concentrations were also compared to previous studies of caffeine testing in other water sources. While only small amounts are being detected at present, there is concern for the future of our caffeinated society.

DNA replication is a vital process ensuring the passing down of intact genetic information over generations. DNA replication in the model organism, Escherichia coli (E. coli), has been extensively studied, providing significant insights into the various enzyme mechanisms. However, while the prime functions of individual components of the replisome have been deciphered, the more complex interactions between the components is an active area of study to understand replisome coupling. At the core of the replisome, the DnaB helicase unwinds the double strand DNA which is followed by rapid synthesis of nascent strand by Polymerase III core (Pol III core). It is proposed that the Tau subunit of the clamp loader complex acts as the physical link between both DnaB and Pol III core, leading to a coupling phenomenon between unwinding and synthesis. Using a reconstituted E. coli replisome in vitro, unwinding of DNA substrates with variable lengths and assemblies can be monitored through electrophoresis to better understand the kinetics of coupling. Mutations have been introduced in the exterior surface of DnaB, disrupting its charged interactions with the excluded strand, and promoting more constricted conformation that result in faster unwinding. The unwinding kinetics of wild‐type versus mutant DnaB enzymes on different length substrates are being examined. Further experiments include Pol III synthesis alone or with proposed coupling from the tau‐subunit and can simultaneously examine unwinding and synthesis. Mutations that affect DnaB conformation or disrupt Pol III‐tau interactions will be utilized to assess their relative influences in maintaining a coupled replisome. Conducting in vitro experiments will provide mechanistic insight into the nature of interactions between these replisome components in a coordinated system. Coupling interactions are also being investigated in vivo using precise CRISPR‐Cas9 genome editing. Coupling mutations have been introduced in the dnaX gene encoding the tau‐subunit. These mutant strains exhibit a reduced growth rate when compared to wild‐type strains, indicating that disrupted coupling leads to reduced doubling times. These strains are being further characterized to determine the genomic and cellular consequences of helicase‐polymerase decoupling in vivo. Therefore, a combination of in vitro and in vivotechniques are being used to better understand the importance of coupling during DNA replication to maintain a stable genome.