Pre-steady state analysis of the assembly of wild type and mutant circular clamps of Escherichia coli DNA polymerase III onto DNA.
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 clamp loading pathway in real time, using pre-steady state fluorescent depolarization measurements to investigate the loading reaction and ATP requirements for the assembly of beta onto DNA. Two beta dimer interface mutants, L273A and L108A, and a nonhydrolyzable ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), 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 reaction. In the presence of ATP and gamma complex, the beta mutants associate with DNA as efficiently as wild type beta. However, completion of the reaction is much slower with the beta mutants because of decreased ATP hydrolysis by the gamma complex, resulting in a much slower release of the mutants onto DNA. The effects of mutations in the dimer interface were similar to the effects of replacing ATP with ATPgammaS 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 completion of the assembly process requires ATP hydrolysis for turnover of the catalytic clamp loader.
Bertram
J G
JG
Department of Biological Sciences, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, USA.
Bloom
L B
LB
Turner
J
J
O'Donnell
M
M
Beechem
J M
JM
Goodman
M F
MF
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D019943:Amino Acid Substitution; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004258:DNA Polymerase III / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005454:Fluorescence Polarization; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D004269:DNA, Bacterial; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III