Mechanism of Action of Ribonucleic Acid-directed Deoxyribonucleic Acid Polymerase

Abstract The synthesis of DNA complementary to rabbit globin mRNA by the enzyme RNA-directed DNA polymerase (from avian myeloblastosis virus) has been studied. The reaction is dependent on mRNA as template, oligo(dT) as primer, and the four deoxynucleoside triphosphates as substrates. The 28 S, 18 S, and 4 S RNA are not effective templates; oligo(dG), oligo(dC), and oligo(dA) are not effective primers. The product of the reaction is a DNA-RNA hybrid, the DNA of which has an electrophoretic mobility of 7 to 10 S. The DNA hybridizes with globin 10 S mRNA but not with 28 S, 18 S, or 4 S RNA, nor does it hybridize with an 8 to 12 S RNA fraction isolated from rabbit liver polysomes. Thus, the RNA-directed DNA polymerase is capable of synthesizing a DNA molecule complementary to globin mRNA; however, based on its size the product does not appear to be a complete copy of the RNA template. Using rabbit reticulocyte 10 S globin mRNA or rabbit liver mRNA as template the RNA-directed DNA polymerase can synthesize poly(dT) when dTTP is the only substrate present; no reaction occurs in the presence of any other single deoxynucleoside triphosphate. A high molecular weight poly(dT) polymer is formed, presumably transcribed from the poly(A) region of the mRNA. Thus, under some conditions, the enzyme may slip during transcription so that parts of the RNA are transcribed more than once in the synthesis of a single DNA molecule.