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 gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 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 approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.
Bertram
J G
JG
Department of Biological Sciences and Chemistry, Hedco Molecular Biology Laboratories, University of Southern California, Los Angeles, California 90089-1340, USA.
Bloom
L B
LB
Hingorani
M M
MM
Beechem
J M
JM
O'Donnell
M
M
Goodman
M F
MF
D000255:Adenosine Triphosphate / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D004258:DNA Polymerase III / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism*; D020035:Holoenzymes / Q000737:chemistry*; D006868:Hydrolysis
D020035:Holoenzymes; D000255:Adenosine Triphosphate; D004247:DNA; D004258:DNA Polymerase III