Mechanism of bacteriophage T4 DNA holoenzyme assembly: the 44/62 protein acts as a molecular motor.
The role of ATP hydrolysis by the 44/62 protein in formation of the stable holoenzyme DNA replication complex has been further elucidated by specifically examining the role that the 44/62 protein plays in loading the 45 protein onto the DNA substrate. A stable phospho-45 protein or phosphorylated holoenzyme complex was not detected or isolated, suggesting that the 44/62 protein may not act as a protein kinase. Product and dead-end inhibition data are consistent with an ordered kinetic mechanism with respect to product release in which phosphate is released from the 44/62 protein prior to ADP. Positional isotope effect studies support this mechanism and failed to demonstrate that ATP hydrolysis by the 44/62 protein is reversible. Steady-state ATPase assays using aluminum tetrafluoride as an inhibitor are also consistent with release of ADP being partially rate-limiting. Aluminum tetrafluoride acts to trap ADP on the enzyme after turnover, forming a stable transition state analog that dissociates slowly from the enzyme. Processive DNA synthesis does not occur using the accessory proteins in the presence of pre- or post-hydrolysis analogs of ATP nor in the presence of ADP-AlF4, indicating that turnover of the 44/62 protein is absolutely required for formation of the holoenzyme complex. Collectively, data obtained regarding ATP hydrolysis by the 44/62 protein are described in terms of the clamp loading protein functioning as a molecular motor, similar to other systems including myosin and kinesin.
Berdis
A J
AJ
Department of Chemistry, The Pennsylvania State University, University Park 16802-6300, USA.
Benkovic
S J
SJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology; D017122:Bacteriophage T4 / Q000201:enzymology*; D002850:Chromatography, Gel; D003067:Coenzymes / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005459:Fluorides / Q000494:pharmacology; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism*
D017607:Aluminum Compounds; D003067:Coenzymes; D004791:Enzyme Inhibitors; D010710:Phosphates; D015534:Trans-Activators; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; C050992:tetrafluoroaluminate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004259:DNA-Directed DNA Polymerase; D005459:Fluorides