| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1997: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
Specific aims : DnaA protein has high affinity for ATP/ADP.Tne ATP-bound form is active for initiation of DNA replication, but the ADP-bound form is not. The first purpose of this work is to reveal mechanism of conformational change of the protein, based on identification of amino acid residue responsible for the ATP-binding. Especially, identification of the residue interacting with phosphate in the gamma position of ATP is most important to know the activation mechanism of the protein. The second purpose is to identify the protein's domain to regulate its activity. For this, dnaA mutants that show lethality for cells bearing the wild-type allele, due to occurrence of excessive initiations will be isolated and biochemical analyzes of the mutant proteins will be performed.
Results and Discussion : Affinity labeling using an ATP-analog that contains modified phosphate at the gamma position and determination of amino acid residue that is covalently bound to this analog suggested that Lys-415 residue interacts with the gamma phosphate of ATP.Next, by site-directed mutagenesis this residue was replace with other ones, and such mutant proteins were purified. Analyzes of these suggested that Lys residue is necessary for activation of the protein by ATP (manuscript in preparation). In the second project, by introducing random mutation in the dnaA cistron, we obtained three dnaA mutant genes that show lethality for cells-bearing the wild-type allele. Sequencing indicated that mutations appear in a distinct region between the ATP-binding domain and the DNA-binding domain of DnaA protein. Purification and characterization of a mutant DnaA isolated here revealed that this mutant protein lacks affinity for ATP but sustains replication activity. These results indicate an importance of conformation of this distinct region for regulation of DnaA function, being a critical finding in study of structure function relationship of the protein.
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