AN ATP-INHIBITED ENDONUCLEASE FROM CAULIFLOWER (BRASSICA-OLERACEA VAR. BOTRYTIS) INFLORESCENCE - PURIFICATION AND CHARACTERIZATION

In our studies on the role of enzymes in plant DNA replication, recomb ination, and repair, we isolated from cauliflower (Brassica oleracea L . var. botrytis) inflorescences a single-stranded DNA-specific endonuc lease that was inhibited by ATP. The endonuclease, designated cauliflo wer nuclease II, was purified to near homogeneity through six successi ve column chromatographies. The enzyme is a single polypeptide with a molecular mass of 70 kDa as judged by the results of sodium dodecyl su lfate-polyacry amide gel electrophoresis, activity gel, and gel-filtra tion column chromatography. The enzyme can cleave a linear or a circul ar single-stranded DNA but cannot cut or nick a double-stranded DNA. T he mode of activity of the nuclease is endonucleolytic and non-process ive. Interestingly, the endonuclease activity is strongly inhibited by less than 0.1 mM ATP, although the role of this inhibition is thus fa r unclear. While ATP gamma S and GTP can also inhibit the activity, ot her ribonucleoside triphosphates are much less effective. The optimum pH of the enzyme is 5.6. The enzyme requires an exceptionally high ion ic strength, 0.2 M KCI for optimum activity, and without these ions no activity can be detected. The endonuclease activity is stimulated by Ca2+, which cannot be replaced by Mg2+ or Mn2+. The features of the en zyme and its relation to plant DNA metabolism are discussed.