DNA filter retention assay for exonuclease activities. Application to the analysis of processivity of phage T5 induced 5'-exonuclease.
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.
D004279:DNA, Viral; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology*; D005374:Filtration / Q000379:methods; D007700:Kinetics; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology*; D013816:Thermodynamics
D004279:DNA, Viral; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I