DISSECTING THE ORDER OF BACTERIOPHAGE-T4 DNA-POLYMERASE HOLOENZYME ASSEMBLY

Most biological organisms rely upon a DNA polymerase holoenzyme for pr ocessive DNA replication. The bacteriophage T4 DNA polymerase holoenzy me is composed of the polymerase enzyme and a clamp protein (the 45 pr otein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loa ded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is inv estigated using a combination of rapid-quench and stopped-flow fluores cence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied p rovided data indicating that the rate-limiting step in holoenzyme asse mbly is associated with the clamp loading process. Pre-steady-state an alysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading p rocess in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, us ing a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 pro tein interacts with the 44/62 complex leading to the hydrolysis of 2 e quiv of ATP, and upon contacting DNA, the remaining two ATP molecules bound to the 44/62 complex are hydrolyzed. Once all four ATP molecules are hydrolyzed, the 45 protein is poised on DNA for association with the polymerase to form the holoenzyme.