Studies on the Synthesis of Deoxyribonucleic Acid by Mammalian Enzymes
II. AN INVESTIGATION OF THE PRIMER REQUIREMENTS OF PARTIALLY PURIFIED REGENERATING RAT LIVER DEOXYRIBONUCLEIC ACID NUCLEOTIDYLTRANSFERASE
Roger Mantsavinos 1 and Benjamin Munson 1
From the
1 From the Department of Biochemical Pharmacology, School of Pharmacy, and the Department of Biochemistry, School of Medicine, State University of New York at Buffalo, Buffalo, New York 14214
A study has been made of the deoxyribonucleic acid primer requirements of partially purified deoxyribonucleic acid deoxynucleotidyltransferase fractionated from regenerating liver of the adult rat. Native, double stranded DNAs derived from either viral, bacterial, or mammalian sources serve more effectively as primers than either single stranded or heat-denatured DNAs for the incorporation of deoxyribonucleoside 5'-triphosphates into polydeoxyribonucleotides. The highly ordered, helical, alternating, copolymer consisting of deoxyadenylate and deoxythymidylate residues was found to be the best primer tested. Destruction of the secondary structure of native DNA primers by either controlled acid or alkaline denaturation or by thermal means decreases priming activity. Additionally, changes in the primary structure of native DNA primers by limited enzymic treatment also altered their priming abilities. Crystalline bovine pancreatic deoxyribonuclease, which cleaves DNA to yield 3'-hydroxyl and 5'-phosphoryl end groups, produced stimulation in priming activity, whereas bovine spleen and micrococcal endonucleases, which produce 3'-phosphoryl and 5'-hydroxyl termini, decrease the priming activity of native DNA primers. Heating and rapid cooling of the primers partially degraded enzymically results in a further reduction of priming activity.
Submitted on November 8, 1965
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