Helicase-driven unwinding defines the architecture of the leading-strand replisome

DNA replication is a fundamental biological process that requires the coordinated activities of DNA helicase, DNA polymerase, and accessory proteins within the replisome. Although helicase-driven unwinding (HDU) has long been the accepted model for replisome progression, recent structural studies have proposed an alternative polymerase-driven unwinding (PDU) model, in which DNA polymerase physically separates duplex DNA. Here, we perform comprehensive structural and biochemical analyses to define the molecular architecture and functional interactions of the bacteriophage T7 leading-strand replisome to distinguish between these two mutually exclusive models. We show that the T7 DNA primase-helicase is positioned directly at the replication fork junction to drive DNA unwinding, whereas T7 DNA polymerase, in complex with its processivity factor E. coli thioredoxin, trails 11 to 15 nucleotides behind. Mutational analysis of T7 DNA polymerase reveals that a {beta}-hairpin previously implicated as a strand-separation pin in PDU functions instead to stabilize DNA polymerase binding to the DNA template. Furthermore, we identify an essential interaction between an acidic amino acid patch in the exonuclease domain of T7 DNA polymerase and the primase domain of T7 DNA primase-helicase, which promotes highly processive leading-strand synthesis and is compatible only with the HDU model. Together, these findings provide evidence supporting helicase-driven unwinding and clarify the molecular organization of the T7 leading-strand replisome. Because the phage T7 replisome serves as a model for DNA replication, these results have broad mechanistic implications for replisomes across taxa. Significance StatementProcessive DNA replication requires coordination between enzymes that unwind and copy DNA. Two mutually exclusive models have been proposed for replisome progression. In helicase-driven unwinding (HDU), DNA helicase separates the parental duplex at the replication fork to generate single-stranded templates for DNA synthesis. In contrast, an alternative model proposes polymerase-driven unwinding (PDU). Using structural and biochemical analyses, we investigated the architecture and functional interactions of the leading-strand replisome from bacteriophage T7. Our results show that DNA helicase performs DNA unwinding, while DNA polymerase follows to synthesize the nascent strand. We identify specific intermolecular interactions essential for leading-strand replication that are compatible only with the HDU model. These findings support helicase-driven unwinding and provide insights with broad mechanistic implications across taxa.