Close‐up of a replisome

Abstract

The assembly of proteins, the replisome, that replicates DNA is comprised of molecular motors and switches that coordinate reactions through multiple interactions. Although differing in complexity, the basic machinery of all replication systems is remarkably conserved throughout evolution. The economy of proteins found in the replication system of bacteriophage T7 has made possible the analysis of a replisome at the molecular level. Four proteins of known crystal structure are sufficient for the basis reactions: T7 DNA polymerase and its processivity factor (E. coli thioredoxin), T7 helicase-primase, and T7 ssDNA binding protein. A reconstituted replisome effects DNA synthesis in which leading and lagging strand synthesis are coordinated and processive. Nascent Okazaki fragments are found within a loop of lagging strand DNA. Occupancy of the nucleotide binding sites of the hexameric helicase, located between adjacent subunits, modulate DNA binding via conformational changes. The transfer of ssDNA from subunit to subunit is coupled to the sequential hydrolysis of dTTP. The recycling of the lagging strand DNA polymerase requires multiple steps and interactions. Interactions between the helicase and primase domains result in the pausing of leading strand synthesis observed during primer synthesis in single molecule experiments. Thioredoxin binds to a unique 76 amino acid fragment located in the thumb sub-domain of T7 DNA polymerase initiating conformational changes that lead to interactions with the primer-template, the helicase-primase, and the ssDNA binding protein. The acidic C-termini of the latter two proteins are essential for these interactions. Thus a three-way switch mediates the interactions of the multiple components during DNA synthesis. Supported by USPHS Grant GM 54397.