Purification, cloning, and characterization of the CEL I nuclease

CEL I, isolated from celery, is the first eukaryotic nuclease known that cl eaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg2+ and Zn2+ for activity, with a pH o ptimum at neutral pH. We have purified CEL I 33 000-fold to apparent homoge neity. A key improvement is the use of alpha-methyl-mannoside in the purifi cation buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatu red, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifi es a new family of neutral pH optimum, magnesium-stimulated, mismatch duple x-recognizing nucleases, within the S1 superfamily.