A mammalian nicking endonuclease

Abstract

Purification and properties are described for an endonuclease isolated from calf thymus which attacks double-stranded, unmodified DNA, primarily by making single-strand breaks. No detectable acid-soluble products arise from the reaction. Double-strand breaks may occasionally be produced by the introduction of single-strand breaks on opposite strands in close proximity. The enzyme does not attack denatured DNA and is not inhibited by tRNA. Added divalent cations are not required for activity, but the enzyme is inhibited by EDTA, which suggests an essential role for bound cations. The reaction is inhibited by Ca2+. The endonuclease has a broad pH optimum and is inactivated by preincubation at temperatures of 45.degree. C and higher. The molecular weight as determined by gel chromatography is about 30,000. Analysis of the products of reaction on the defined substrate, bacteriophage T3 DNA, by sedimentation in alkaline sucrose density gradients indicates limit products with chain lengths of about 0.8 .times. 106 daltons. Electrophoresis in agarose gels shows these products to be heterogeneous in size. The endonuclease appears to generate 3''-hydroxyl and 5''-phosphate ends. The ability of the endonuclease to utilize bovine DNA as substrate argues against a restriction role for this enzyme.