Molecular Contacts and Kinetic Control within the Replisome maintain Coupled DNA Unwinding and Synthesis

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Accurate and faithful duplication of the genome requires rapid, efficient, and coordinated activities of a collection of proteins within the replisome complex. DNA unwinding followed by DNA synthesis keep the replisome coupled and protected. Here, using a combination of intricate whole replisome unwinding and synthesis experiments on rolling circle amplification substrates, we investigate the specific molecular contacts, and kinetic control used by the bacterial replisome to maintain replisome coupling and prevent genomic instability. The hexameric helicase that performs DNA unwinding is at the forefront of the replisome providing a platform for all other proteins in the replisome complex. We have created and validated site-specific mutants of Escherichia coli (E. coli) DnaB helicase that show rapid unwinding as single enzymes on a short DNA substrate. When these same DnaB mutants are used in a complete in vitro replication system on a long DNA substrate, unwinding increased, and interestingly, the leading strand synthesis rate and the abundance of DNA product decreased. Therefore, the data suggest an extensive helicase-polymerase decoupling of the replisome. Identifying and validating these dynamic molecular contacts and kinetics required for coupled DNA unwinding and synthesis can provide basis for further studies in other Domains of life.