1988 Fiscal Year Final Research Report Summary
Reaction Mechanism of ATP-dependent DNase and its Role in the genetic Recombination Process in the Cell.
| Project/Area Number |
60440105
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Fujita-Gakuen Health University, Medical School |
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Principal Investigator |
TAKAGI Yasuyuki Eujita-Gakuen Health University, Medical School, 医学部, 教授 (50037313)
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| Project Period (FY) |
1985 – 1988
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| Keywords | ATP-dependent DNase / recBC Enzyme / Genetic Recombination / 細胞内遺伝子組換え / ATP |
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| Research Abstract |
ATP-dependent DNase is widely distributed among various microorganisms, and catalyzes the exonucleolytic degradation of lineal double and single stranded DNA in the presence of added ATP, producing the oligonucleotides consisted of about 5 nucleotides. Meanwhile mutations in the Escherichia coli recB and recC genes are known to lead to a mutant phenotype, reduced recombination and increased sensitivity to ultraviolet-light irradiation or mitomycin C treat-ment. Both genes are also found to control the production of the ATP-dependent dnase in E. coli cells. Therefore in this research project, it was aimed to isolate both ganes and purify the gene products in large scale, then to analyze the reaction mechanism in detail.
The E. coli thyA, recC, recB and argA gene region was cloned in a cosmid, and the recB and recC genes were separately subcloned, proving that these two genes consist of independent cistrons. The base sequences of both genes were analyzed, and it was confirmed that the rec
… More
B gene encodes a protein of 1180 amino acids, and the recC gene 1122 amino acids.
Then the properties of the recB and recC gene products were studied using recB and recC gene-inserted plasmids. recB mutants and recC mutants lacked ATP-dependent DNase, but showed apar-ent recovery of enzyme activity on introduction of plasmids carrying the recB and recC gene, respectively. The ATP-dependent DNase was also constructed in vitro by mixing the recB and recC gene products by the plasmids with the corresponding gene. Specific labeling of plasmid-encoded proteins by the maxicell method showed that the recB and recC gene products were 135,000 and 125,000 dalton protein, respectively. However a possibility that the addition of another gene product is requested to construct the whole enzyme was suggested.
Finally it was found that ATP-dependent DNase of Bacillus laterosporus consumes 3 ATPs at pH 8.3, and 2.2 at 6.3 per every one phosphodiester bond-cleavage, suggesting to catalyze the reaction of two types. Less
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