Jg. Bertram et al., PRE-STEADY-STATE ANALYSIS OF THE ASSEMBLY OF WILD-TYPE AND MUTANT CIRCULAR CLAMPS OF ESCHERICHIA-COLI DNA-POLYMERASE-III ONTO DNA, The Journal of biological chemistry, 273(38), 1998, pp. 24564-24574
The beta protein, a dimeric ring-shaped clamp essential for processive
DNA replication by Escherichia coli DNA polymerase III holoenzyme, is
assembled onto DNA by the gamma complex. This study examines the clam
p loading pathway in real time, using pre-steady state fluorescent dep
olarization measurements to investigate the loading reaction and ATP r
equirements for the assembly of beta onto DNA. Two beta dimer interfac
e mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosin
e 5'-O-(3-thiotriphosphate) (ATP gamma S), have been used to show that
ATP binding is required for gamma complex and beta to associate with
DNA, but that a gamma complex-catalyzed ATP hydrolysis is required for
gamma complex to release the beta DNA complex and complete the reacti
on. In the presence of ATP and gamma complex, the beta mutants associa
te with DNA as efficiently as wild type beta. However, completion of t
he reaction is much slower with the beta mutants because of decreased
ATP hydrolysis by the gamma complex, resulting in a much slower releas
e of the mutants onto DNA. The effects of mutations in the dimer inter
face were similar to the effects of replacing ATP with ATP gamma S in
reactions using wild type beta. Thus, the assembly of beta around DNA
is coupled tightly to the ATPase activity of the gamma complex, and co
mpletion of the assembly process requires ATP hydrolysis for turnover
of the catalytic clamp loader.