Abstract

A new purification procedure for Escherichia coli DNA polymerase yields about 10 mg of homogeneous enzyme per kg of cell paste, with exonuclease III as a by-product. DNA polymerase consists of a single polypeptide chain of molecular weight 1.09 × 105. This tentative conclusion is based on (a) an unchanged molecular weight after unfolding in solutions containing guanidine hydrochloride and mercaptoethanol, (b) the presence of approximately one residue of NH2-terminal methionine, and (c) the presence of a single zone on polyacrylamide gel electrophoresis in a denaturing solvent at several pH values. The enzyme contains one disulfide bond and a single reactive sulfhydryl group, which can be modified by iodoacetic acid without change in either polymerase or its associated exonuclease activity. There is less than one phosphorus atom per molecule, thus minimizing the possibility of enzyme-associated nucleotide material in stoichiometric amounts.