The clamp loader of Escherichia coli DNA polymerase III: Kinetics of the ATP-dependent steps in the sliding -clamp loading reaction

DNA polymerase III holoenzyme, the principal enzyme responsible for E. coli chromosomal replication, synthesizes stretches of DNA thousands of nucleotides long at a rate approaching 750 nucleotides s-1 without dissociation. A ring-shaped DNA sliding-clamp "beta" topologically links the polymerase to DNA. A clamp loader, "gamma complex", assembles beta on DNA in an ATP-dependent reaction. This dissertation project was undertaken for investigation of the mechanism of beta clamp loading by the clamp loader for processive replication. ATP binding and hydrolysis activities of the clamp loader promote conformational changes modulating its binding affinity for the clamp and DNA. Using fluorescence-based steady-state and real-time stopped-flow methods, the kinetics of these dynamic conformational changes were measured. Pre-steady-state ATP hydrolysis assays performed in the absence or presence of beta resulted in biphasic or monophasic kinetics respectively. Biphasic kinetics suggested that the clamp loader, equilibrated with ATP, exists in a mixture of two dominant species. Addition of beta converted this mixture into a single activated population, effectively increasing its concentration. These experiments, in addition to adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS)-chase assays showed that activated gamma complex hydrolyzed one ATP per gamma-subunit at a rate faster than ATP dissociation. The hypothesis that gamma complex exists in multiple species with ATP was confirmed by measuring pre-steady-state clamp loading kinetics in DNA-binding assays. When gamma complex was equilibrated with ATP, a mixture of two species formed which when mixed with DNA and beta exhibited biphasic DNA binding kinetics. When equilibrated with ATP and beta, rapid monophasic DNA binding kinetics resulted. Direct mixing of gamma complex with DNA beta and ATP displayed slow monophasic kinetics, limited by the conformational change rate. The rate of the conformational changes separating the two dominant species was determined by investigating their evolution in equilibration time with ATP. Computer modeling of these experimental data revealed a conformational change rate of ∼4.5 s-1. Comparison of the kinetics of a "minimal" clamp loader complex missing chi and psi subunits, revealed that these subunits facilitate the conformational changes in gamma complex required for modulation of beta and DNA binding affinities during the clamp loading reaction.