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J. Biol. Chem., Vol. 275, Issue 37, 28413-28420, September 15, 2000
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From the Escherichia coli DNA polymerase III
holoenzyme is a multisubunit composite containing the
Molecular Mechanism and Energetics of Clamp Assembly in
Escherichia coli
THE ROLE OF ATP HYDROLYSIS WHEN 
COMPLEX LOADS 
ON
DNA*
,
,**
Department of Biological Sciences and
Chemistry, Hedco Molecular Biology Laboratories, University of Southern
California, Los Angeles, California 90089-1340, the
§ Department of Biochemistry and Molecular Biology,
University of Florida, Gainsville, Florida 32610-0245, the
¶ Rockefeller University and Howard Hughes Medical Institute, New
York, New York 10021, and the Department of Molecular Physiology
and Biophysics, Vanderbilt University, Nashville, Tennessee 37232
sliding clamp
and clamp loading complex. The complex requires ATP to load 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 ·
complex composite rapidly binds primer/template DNA in an ATP
hydrolysis independent step. Once bound, two molecules of ATP are
rapidly hydrolyzed (~34 s
1). Following hydrolysis, complex dissociates from the DNA (~22 s1). Once
dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only
~3 s1. Two single-site 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.
*
This work was supported by National Institutes of Health
Grants GM21422 (to M. F. G.), GM55596 (to L. B. Bl.),
GM38839 (M. O'D.), and GM45990 and RR5823 (to J. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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