Multiple ATP binding is required to stabilize the "activated" (clamp open) clamp loader of the T4 DNA replication complex.
Most DNA replication systems include a sliding clamp that encircles the genomic DNA and links the polymerase to the template to control polymerase processivity. A loading complex is required to open the clamp and place it onto the DNA. In phage T4 this complex consists of a trimeric clamp of gp45 subunits and a pentameric loader assembly of four gp44 and one gp62 subunit(s), with clamp loading driven by ATP binding. We measure this binding as a function of input ligand concentration and show that four ATPs bind to the gp44/62 complex with equal affinity. In contrast, the ATPase rate profile of the clamp-clamp loader complex exhibits a marked peak at an input ATP concentration close to the overall Kd (approximately 30 microm), with further increases in bound ATP decreasing the ATPase rate to a much lower level. Thus the progressive binding of the four ATPs triggers a conformational change in the complex that markedly inhibits ATPase activity. This inhibition is related to ring opening by using a clamp that is covalently cross-linked across its subunit interfaces and thus rendered incapable of opening. Binding of this clamp abolishes substrate inhibition of the ATPase but leaves ATP binding unchanged. We show that four ATP ligands must bind to the T4 clamp loader before the loader can be fully "activated" and the clamp opened, and that ATP hydrolysis is required only for release of the loader complex after clamp loading onto the replication fork has been completed.
Pietroni
Paola
P
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403-1229, USA.
von Hippel
Peter H
PH
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D017122:Bacteriophage T4 / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry / Q000494:pharmacology; D004261:DNA Replication; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D007700:Kinetics; D008954:Models, Biological; D008968:Molecular Conformation; D009711:Nucleotides / Q000737:chemistry; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D013379:Substrate Specificity
D003432:Cross-Linking Reagents; D009711:Nucleotides; D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases