A Deoxyribonucleic Acid Phosphatase-exonuclease from Escherichia Coli. I. Purification of the Enzyme and Characterization of the Phosphatase Activity.

A major problem regarding deoxyribonucleic acid polymerase action is the nature of the deoxyribonucleic acid template. Alterations of DNA may reduce or enhance its priming capacity, and both kinds of change have complicated assays of polymerase purification. When extensively purified polymerase was chromatographed on hydroxylapatite, over 90% of the protein and all the activity, as measured with the deoxyadenylate-deoxythymidylate copolymer as primer, were found in a single symmetrical peak. However, the amount of enzyme recovered was less than 20% as determined with calf thymus DNA as primer. Eluted earlier in the chromatogram was a minute protein peak (less than 2% of the total), free of polymerase but capable of restoring the main polymerase peak to full activity with the thymus DNAprimed assay. This new enzyme has been purified extensively from Escherichiu coli extracts and identified as a phosphatase highly specific for a phosphate residue esterified to the 3’-hydroxyl terminus of a DNA chain. Such a 3’-phosphoryl-terminated chain not only fails to serve as primer, but is a potent inhibitor of polymerase action (1). Thus the hydrolytic removal of this 3’-phosphoryl group is essential for the priming function of chains which bear them. The present report deals with the purification of this DNA phosphatase and the specificity of its action. The succeeding paper reveals another aspect of the action of this enzyme, the capacity to cleave 5’-nucleotidyl residues stepwise from the 3’hydroxyl end of a DNA chain. The enzyme thus hydrolyzes phosphomonoor diester bridges between the phosphate and the 3’-hydroxyl group. Given a 3’-phosphoryl terminus on DNA, as shown in Scheme 1, the enzyme releases inorganic phosphate and then proceeds as an esonuclease with the stepwise release of 5’-mononucleotides. The ability of this DNA phosphatase activity to remove the 3’-phosphoryl end groups of high molecular weight oligonucleotides provides a useful reagent for studying the effect of such end groups in the DNA-synthesizing system (1).