Dataset Information

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

ABSTRACT: 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.

SUBMITTER: Anderson SG

PROVIDER: S-EPMC2759312 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Anderson Stephen G SG Thompson Jennifer A JA Paschall Christopher O CO O'Donnell Mike M Bloom Linda B LB

Biochemistry 20090901 36

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 fl ...[more]

PMID: 19663416

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Project description:Extracellular adenosine triphosphate (ATP) regulates pancreatic duct function via P2Y and P2X receptors. It is well known that ATP is released from upstream pancreatic acinar cells. The ATP homeostasis in pancreatic ducts, which secrete bicarbonate-rich fluid, has not yet been examined. First, our aim was to reveal whether pancreatic duct cells release ATP locally and whether they enzymatically modify extracellular nucleotides/sides. Second, we wished to explore which physiological and pathophysiological factors may be important in these processes. Using a human pancreatic duct cell line, Capan-1, and online luminescence measurement, we detected fast ATP release in response to pH changes, bile acid, mechanical stress and hypo-osmotic stress. ATP release following hypo-osmotic stress was sensitive to drugs affecting exocytosis, pannexin-1, connexins, maxi-anion channels and transient receptor potential cation channel subfamily V member 4 (TRPV4) channels, and corresponding transcripts were expressed in duct cells. Direct stimulation of intracellular Ca(2+) and cAMP signalling and ethanol application had negligible effects on ATP release. The released ATP was sequentially dephosphorylated through ecto-nucleoside triphosphate diphosphohydrolase (NTPDase2) and ecto-5'-nucleotidase/CD73 reactions, with respective generation of adenosine diphosphate (ADP) and adenosine and their maintenance in the extracellular medium at basal levels. In addition, Capan-1 cells express counteracting adenylate kinase (AK1) and nucleoside diphosphate kinase (NDPK) enzymes (NME1, 2), which contribute to metabolism and regeneration of extracellular ATP and other nucleotides (ADP, uridine diphosphate (UDP) and uridine triphosphate (UTP)). In conclusion, we illustrate a complex regulation of extracellular purine homeostasis in a pancreatic duct cell model involving: ATP release by several mechanisms and subsequent nucleotide breakdown and ATP regeneration via counteracting nucleotide-inactivating and nucleotide-phosphorylating ecto-enzymes. We suggest that extracellular ATP homeostasis in pancreatic ducts may be important in pancreas physiology and potentially in pancreas pathophysiology.

Dataset Information

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Publications

Temporal correlation of DNA binding, ATP hydrolysis, and clamp release in the clamp loading reaction catalyzed by the Escherichia coli gamma complex.

Similar Datasets

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