A slow ATP-induced conformational change limits the rate of DNA binding but not the rate of beta clamp binding by the escherichia coli gamma complex clamp loader.
In Escherichia coli, the gamma complex clamp loader loads the beta-sliding clamp onto DNA. The beta clamp tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, gamma complex binds beta and DNA to form a ternary complex. Binding to primed template DNA triggers gamma complex to hydrolyze ATP and release the clamp onto DNA. Here, we investigated the kinetics of forming a ternary complex by measuring rates of gamma complex binding beta and DNA. A fluorescence intensity-based beta binding assay was developed in which the fluorescence of pyrene covalently attached to beta increases when bound by gamma complex. Using this assay, an association rate constant of 2.3 x 10(7) m(-1) s(-1) for gamma complex binding beta was determined. The rate of beta binding was the same in experiments in which gamma complex was preincubated with ATP before adding beta or added directly to beta and ATP. In contrast, when gamma complex is preincubated with ATP, DNA binding is faster than when gamma complex is added to DNA and ATP at the same time. Slow DNA binding in the absence of ATP preincubation is the result of a rate-limiting ATP-induced conformational change. Our results strongly suggest that the ATP-induced conformational changes that promote beta binding and DNA binding differ. The slow ATP-induced conformational change that precedes DNA binding may provide a kinetic preference for gamma complex to bind beta before DNA during the clamp loading reaction cycle.
Thompson
Jennifer A
JA
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
Paschall
Christopher O
CO
O'Donnell
Mike
M
Bloom
Linda B
LB
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001665:Binding Sites; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D021122:Protein Subunits
D004269:DNA, Bacterial; D021122:Protein Subunits; D000255:Adenosine Triphosphate; C101139:beta subunit, DNA polymerase III; D004258:DNA Polymerase III