Deoxyribonucleic Acid Polymerase: Two Distinct Enzymes in One Polypeptide

Abstract

Proteolysis of Escherichia coli DNA polymerase (mol wt 109,000) yields two fragments: a large fragment (mol wt 76,000) which retains polymerase and 3′ → 5′ exonuclease activities, and a small fragment (mol wt 36,000) which retains only the 5′ → 3′ exonuclease activity. The large fragment has been obtained in two ways: by cleavage of the intact enzyme with subtilisin or other proteases, and by isolation from E. coli extracts, presumably after proteolysis had occurred in the extract. The large fragment carries out DNA synthesis on nicked double-stranded DNA without degradation of the strand ahead of the growing point, and catalyzes a 3′ → 5′ exonuclease action on both single- and doublestranded DNA with production of nucleoside monophosphates. The large fragment is inactive in unprimed poly[d(A-T)] synthesis, but catalyzes primed poly[d(A - T)] synthesis at a relatively linear rate generating poly[d(A-T)] molecules up to 10 µm in length. The single cysteine residue and the single disulfide group of the intact enzyme are retained in the large fragment as are the binding sites for a deoxyribonucleoside triphosphate and a 3′-hydroxyl ribonucleotide (primer terminus).