Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.
Clamp loaders are multisubunit complexes that use the energy derived from ATP binding and hydrolysis to assemble ring-shaped sliding clamps onto DNA. Sliding clamps in turn tether DNA polymerases to the templates being copied to increase the processivity of DNA synthesis. Here, the rate of clamp release during the clamp loading reaction was measured directly for the first time using a FRET-based assay in which the E. coli gamma complex clamp loader (gamma3deltadelta'chipsi) was labeled with a fluorescent donor, and the beta-clamp was labeled with a nonfluorescent quencher. When a beta.gamma complex is added to DNA, there is a significant time lag before the clamp is released onto DNA. To establish what events take place during this time lag, the timing of clamp release was compared to the timing of DNA binding and ATP hydrolysis by measuring these reactions directly side-by-side in assays. DNA binding is relatively rapid and triggers the hydrolysis of ATP. Both events occur prior to clamp release. Interestingly, the temporal correlation data and simple modeling studies indicate that the clamp loader releases DNA prior to the clamp and that DNA release may be coupled to clamp closing. Clamp release is relatively slow and likely to be the rate-limiting step in the overall clamp loading reaction cycle.
Anderson
Stephen G
SG
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32610-0245, USA.
Thompson
Jennifer A
JA
Paschall
Christopher O
CO
O'Donnell
Mike
M
Bloom
Linda B
LB
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics; D004258:DNA Polymerase III / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis; D008958:Models, Molecular; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C103076:DnaX protein, Bacteria; D029968:Escherichia coli Proteins; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D004258:DNA Polymerase III; C444660:gamma subunit, F(1) ATPase; D006180:Proton-Translocating ATPases