RAPID ASSEMBLY OF THE BACTERIOPHAGE-T4 CORE REPLICATION COMPLEX ON A LINEAR PRIMER TEMPLATE CONSTRUCT

DNA synthesis on a primed DNA substrate by bacteriophage T4 requires t he assembly of a core replication complex consisting of the T4 DNA pol ymerase, a single-stranded binding protein (32 protein), and the acces sory proteins 44/62 and 45. In this paper, we demonstrate the successf ul assembly of this core complex on a short linear primer/template sys tem at levels of accessory proteins equivalent to the concentration of primer 3' ends. The key to this assembly is the presence of streptavi din molecules bound at each end of the DNA substrate via biotin moieti es incorporated into the template strand. Streptavidin serves to block the ends of the primer/template, thus preventing translocation of the accessory proteins away from the site of assembly and their subsequen t dissociation from the ends of the primer/template. Complex assembly on this substrate requires ATP and the presence of both the 44/62 and 45 proteins. The time required for assembly of a full enzyme equivalen t of complex in our system is almost-equal-to 2 s.