Enzymatic Synthesis of Deoxyribonucleic Acid

The purified Escherichia coli DNA polymerase contains a single sulfhydryl group which reacts with Hg(II) to produce either a monomer containing a single mercury atom or a dimer of two protein molecules joined by a mercury atom. Both forms retain full polymerase and exonuclease activities, implying that the sulfhydryl group is relatively exposed and not essential for enzymatic activity. The acylating agent, N-carboxymethylisatoic anhydride, reacts with an observed maximum of 11 sites on the enzyme to form a highly fluorescent derivative with altered functional properties. Only 0.2% of the original polymerase activity but 920% of the exonuclease activity are observed at pH 7.4 with DNA as primer or substrate. Measurements of release of inorganic pyrophosphate and binding of triphosphates indicate that the predominant effect of this acylating agent is to cause a marked reduction in the affinity for deoxyribonucleoside triphosphate substrates, but concomitant changes in the interaction of the enzyme with polynucleotides are probably also involved. The data are discussed in terms of a model in which the several activities of the enzyme are catalyzed at the same active center, which is differentiated into physically distinct sites.