Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases

Abstract

DNA polymerases catalyze the addition of deoxyribonucleotides onto DNA primers in a template-directed manner. The requirement for template instruction distinguishes these enzymes from other nucleotidyl transferases, such as terminal deoxynudeotidyl transferase, that do not utilize a template. An ollgonudeotide substrate was used to characterize a novel, non-templated nudeotide addition reaction carried out by DNA polymerases from a variety of procaryotic and eucaryottc sources. The products of the reaction, in which a deoxyribonucleotide was added to the 3' hydroxyl terminus of a blunt-ended DNA substrate, were analyzed by electrophoresis on high resolution, denaturing polyacrylamide gels. DNA polymerase from Thermus aquaticus. polymerase a from chick embryo, rat polymerase B, reverse transcriptase from avlan myeloblastosis virus, and DNA polymerase I fromSaccharomyces cerevisiae all carried out the blunt-end addition reaction. The reaction required a duplex DNA substrate but did not require coding information from the template strand. These results demonstrate that template instruction is not an absolute requirement for the catalysis of nucleotidyl transfer reactions by DNA polymerases. © 1988 IRL Press Limited, Oxford, England.