Tau Mediated Coupling Interactions between Pol III Core DNA Synthesis and DnaB Helicase Unwinding.

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.