Purification and Characterization of an Endonuclease Specific for Single-Stranded DNA from Bacillus subtilis Marburg

Abstract

Bacillus subtilis Marburg TI (thy, trpC₂) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular fl DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endo-nuclease MU), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57, 000 daltons. The cleavage products have 5'-phosphoryl termini. At low con-centrations, double-stranded DNA is not split to any detectable extent. At high concen-trations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5, 000: 1 by comparing the initial rates of introducing single-strand breaks into the DNA's.