Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system.
Processive DNA replication requires the loading of a multisubunit ring-shaped protein complex, known as a sliding or processivity clamp, onto the primer-template (p/t) DNA. This clamp then binds to the replication polymerase to form a processive polymerase holoenzyme. The processivity of the holoenzyme derives from the topological properties of the clamp, which encircles the DNA without actually binding to it. Multisubunit complexes known as clamp-loaders utilize ATP to drive the placement of this ring around the DNA. To further understand the role of ATP binding and hydrolysis in driving clamp-loading in the DNA replication system of bacteriophage T4, we report the results of a series of presteady-state and steady-state kinetic ATPase experiments involving the various components of the reconstituted system. The results obtained are consistent with a mechanism in which a slow step, which involves the binary ATP-bound clamp-clamp loader complex, activates this complex and permits p/t DNA to bind and stimulate ATP hydrolysis. ATP hydrolysis itself, as well as the subsequent (after clamp-loading) dissociation of the clamp-loader and the slippage of the loaded clamp from the p/t DNA construct, are shown to be fast steps. A second slow step occurs after ATP hydrolysis. This step involves the dissociated clamp loader complex and may reflect ADP release. Only one molecule of ATP is hydrolyzed per clamp-loading event. Rate constants for each step, and an overall reaction mechanism for the T4 clamp-loading system, are derived from these data and from other results in the literature. The principles that emerge fit into a general framework that can apply to many biological processes involving ATP-driven reaction cycles.
Pietroni
P
P
Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403-1229, USA.
Young
M C
MC
Latham
G J
GJ
von Hippel
P H
PH
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D017122:Bacteriophage T4 / Q000201:enzymology* / Q000235:genetics*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D011485:Protein Binding; D021122:Protein Subunits; D012313:RNA / Q000235:genetics; D013698:Templates, Genetic; D017186:Titrimetry; D015534:Trans-Activators / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
D020035:Holoenzymes; D046911:Macromolecular Substances; D021122:Protein Subunits; C034477:RNA primers; D015534:Trans-Activators; D014764:Viral Proteins; C074855:gene 44 protein, Enterobacteria phage T4; C074555:gene 45 protein, Enterobacteria phage T4; C088659:gp62 protein, bacteriophage T4; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases