Purification and Properties of a Manganese-dependent Endonuclease From Tobacco Cell Cultures

Abstract

Summary From suspension cultured cells of Nicotiana tabacum a manganese-dependent DNA endonuclease was purified to homogeneity with a molecular weight of 24,000 in SDS gel electrophoresis. It has a pH optimum of 7.0 and a temperature optimum of 37°C. The enzyme does not digest RNA or synthetic polyribonucleotides; it has no exonucleolytic and nucleotidase activity and is inhibited by nucleoside triphosphates in the mM range. The tobacco endonuclease cleaves heat denatured DNA slightly faster than native DNA; however, in both cases the products are large fragments 3 to 4 kb in size. It is shown with native DNA, that the mode of action of the enzyme is to generate single-strand nicks probably at AT-rich clusters. Circular superhelical DNA of pBR 322 is digested via the relaxed ring-open to the linear form followed by the production of large fragments. With both E. coli and pBR 322 DNA the fragments obtained are double-stranded with single-stranded ends which can be further trimmed by nuclease S1. The present enzyme differs from the endonuclease recently purified from nuclei of the same source (Szopa et al., 1983) by its molecular weight, the metal ion-dependency, pH-optimum, inhibition by ATP and the mode of action especially with superhelical DNA. The present enzyme is copurified with RNA-polymerase I.