The Terminal Nucleotidyltransferase of Calf Thymus Nuclei

Abstract

Abstract An enzyme from calf thymus nuclei which incorporates ribonucleotides or deoxyribonucleotides into terminal positions of deoxyribonucleic acid has been purified and its mechanism of action elucidated. Distinctions between this enzyme and replicative deoxynucleotidyltransferase (DNA polymerase), the terminal deoxynucleotidyltransferase of calf thymus cytoplasm, and other polymerases have been established. The enzyme utilizes a nucleoside triphosphate precursor and a denatured deoxyribonucleic acid primer in a reaction which requires Mg++, ethylenediaminetetraacetic acid, and a sulfhydryl compound for maximal activity. No nucleotide specificity in the deoxyribonucleic acid acceptor can be demonstrated other than that the addition of ribonucleotide to ribonucleotide does not occur. The same DNA acceptor sites appear to be involved in the incorporation of all the nucleoside triphosphates tested. Oligodeoxythymidylic acid, three nucleotides in length, cannot function as a primer in this system. The terminal β- and γ-phosphates of the nucleoside triphosphate substrates are liberated as free pyrophosphate during the reaction. No exchange between free pyrophosphate and a nucleoside triphosphate occurs. The reaction thus appears to be irreversible. The same enzyme from calf thymus nuclei is apparently responsible for the incorporation of cytidine triphosphate, guanosine triphosphate, adenosine triphosphate, and deoxyadenosine triphosphate. Differences in the incorporation rates of these substrates are shown. Under the appropriate incubation conditions, this enzyme may be used as a preparative tool for the synthesis of DNA bearing a high percentage of ribonucleotide termini. Such DNA has been utilized in an assay for exonuclease activity.