Molecular mechanism and energetics of clamp assembly in Escherichia coli -The role of ATP hydrolysis when gamma complex loads beta on DNA

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamm a complex requires ATP to load beta onto DNA. A two-color fluorescence spec troscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydro lysis (green fluorescence; phosphate binding protein assay) were simultaneo usly measured with millisecond timing resolution. The two temporally correl ated stopped-flow signals revealed that a preassembled beta.gamma complex c omposite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (similar to 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA (si milar to 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only similar to 3 s(-1). Two single-site beta dimer interface mutants w ere examined which had impaired steady-state rates of ATP hydrolysis. The p re-steady-state correlated kinetics of these mutants revealed a pattern ess entially identical to wild type. The anisotropy data showed that these muta nts decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.