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.
DiIorio, M. C., Kulczyk, A. W.
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