DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease
| Autor: | Martine Joannes, Alain Jacquemin-Sablon, Jean Marie Saucier |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
Exonuclease
DNA polymerase II Biochemistry Bacteriophage chemistry.chemical_compound Escherichia coli Nucleotide Klenow fragment Gel electrophoresis chemistry.chemical_classification biology Phosphoric Diester Hydrolases Processivity biology.organism_classification Molecular biology Kinetics chemistry Phosphodiesterase I Enzyme Induction DNA Viral biology.protein Biophysics Thermodynamics T-Phages DNA Filtration |
| Zdroj: | Biochemistry. 24(27) |
| ISSN: | 0006-2960 |
| Popis: | The 5'-exonuclease of phage T5 has been purified nearly to homogeneity by using a simple and fast procedure. The kinetic properties of the purified enzyme have been studied by using a new sensitive assay based upon retention by nitrocellulose filters of DNA with short protruding single-stranded ends. The enzyme is specifically stimulated by KCl. Its Km is 2.2 X 10(-7) M at 30 degrees C, and its turnover number is 0.33 DNA molecule transformed per minute. The filter retention assay shows that the T5 exonuclease acts by a semiprocessive mechanism, removing from DNA ends about 30 nucleotides on the average per cycle. The degree of enzyme processivity increases with increasing magnesium concentrations. |
| Databáze: | OpenAIRE |
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