Dissecting the order of bacteriophage T4 DNA polymerase holoenzyme assembly.
Most biological organisms rely upon a DNA polymerase holoenzyme for processive DNA replication. The bacteriophage T4 DNA polymerase holoenzyme is composed of the polymerase enzyme and a clamp protein (the 45 protein), which functions as a processivity factor by strengthening the interaction between DNA and the holoenzyme. The 45 protein must be loaded onto DNA by a clamp loader ATPase complex (the 44/62 complex). In this paper, the order of events leading to holoenzyme formation is investigated using a combination of rapid-quench and stopped-flow fluorescence spectroscopy kinetic methods. A rapid-quench strand displacement assay in which the order of holoenzyme component addition is varied provided data indicating that the rate-limiting step in holoenzyme assembly is associated with the clamp loading process. Pre-steady-state analysis of the clamp loader ATPase activity demonstrated that the four bound ATP molecules are hydrolyzed stepwise during the clamp loading process in groups of two. Clamp loading was examined with stopped-flow fluorescence spectroscopy from the perspective of the clamp itself, using a site-specific, fluorescently labeled 45 protein. A mechanism for T4 DNA polymerase holoenzyme assembly is proposed in which the 45 protein interacts with the 44/62 complex leading to the hydrolysis of 2 equiv 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.
Sexton
D J
DJ
Department of Chemistry, The Pennsylvania State University, University Park 16802, USA.
Kaboord
B F
BF
Berdis
A J
AJ
Carver
T E
TE
Benkovic
S J
SJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D017122:Bacteriophage T4 / Q000201:enzymology*; D003067:Coenzymes / Q000737:chemistry / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003067:Coenzymes; D015534:Trans-Activators; D014764:Viral Proteins; C074555:gene 45 protein, Enterobacteria phage T4; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases