DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease.
Biochemistry, 1985/12/31;24(27):8043-9.
Joannes M, Saucier JM, Jacquemin-Sablon A
PMID: 3004572
Impact factor: 3.321
Abstract
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.
MeSH terms
DNA, Viral; Enzyme Induction; Escherichia coli; Filtration; Kinetics; Phosphodiesterase I; Phosphoric Diester Hydrolases; T-Phages; Thermodynamics
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