DNA Polymerase‐Parental DNA Interaction is Essential for Helicase‐Polymerase Coupling in Bacteriophage T7 DNA Replication

Abstract

DNA helicase and polymerase work cooperatively at the replication fork to unwind the parental DNA and perform leading strand DNA synthesis. It was believed that the helicase migrates at the forefront of the replication fork and unwinds DNA to provide a template for DNA polymerase. Although extensively investigated, the molecular basis of the helicase‐polymerase coupling is not fully understood. Replisome from bacteriophage T7 has been a model system for estimating mechanisms of DNA replication. Recently, the first structure of the helicase‐polymerase complex at a replication fork was reported with the T7 system. The T7 replisome structure suggested that the helicase and polymerase sandwich the parental DNA, with each enzyme pulling one daughter strand in opposite directions. Interestingly, it is the T7 polymerase but not helicase that carries the parental DNA with a β‐hairpin loop stacking at the fork opening and a positively charged cleft that holds the parental DNA. However, the role of the polymerase‐parental DNA interaction in helicase‐polymerase coupling has not been investigated. Here, we created and characterized T7 polymerases with perturbed β‐hairpin loop and the positively charged cleft. Mutations on both structural elements significantly reduced the strand displacement synthesis by T7 polymerase but had a minor effect on DNA synthesis against linear DNA substrate. Moreover, the mutations eliminate synergistic helicase polymerase binding to the DNA fork substrate and replication fork progression in mobility shift assay and rolling‐circle replication assays. Our biochemical data suggested that polymerase plays a dominant role in helicase‐polymerase coupling and replisome progression.