Purification and properties of RNA-dependent DNA polymerase from cytoplasmic A-type particles of murine mammary tumor virus.
1. The RNA-dependent DNA polymerase associated with cytoplasmic A-type particles of murine mammary tumor virus was isolated to near homogeneity by a procedure which includes dissociation of proteins from RNA by centrifugation in a step gradient of cesium chloride, followed by an affinity chromatography on poly(rC)-agarose column. Two species of DNA polymerase were separated by the chromatography: enzyme I in 0.55 M NaCl and enzyme II in 0.80 M NaCl eluate, respectively. 2. The purified DNA polymerases consist of two major polypeptides, with molecular weights of 94,000 and 42,000, as the intrinsic subunits. Both enzyme protomers with a sedimentation coefficient of 6.3--6.4 S and a molecular weight of 115,000--120,000 associate to form active oligomers in low-ionic-strength buffer. 3. Both enzymes catalyzed the hydrolysis of RNA in RNA . DNA hybrids as well as the RNA-dependent synthesis of DNA; these are the intrinsic activities of the reverse transcriptase from B-type particles of murine mammary tumor virus as well as from avian and mammalian C-type oncornaviruses. The general catalytic properties are similar to those of the enzyme from B-type particles. Compared with DNA polymerases I, DNA polymerase II exhibited a high affinity for all the template-primers tested and, in addition, a high preference for (rC)N . (dG)12--18.
D000818:Animals; D004256:DNA Polymerase I / Q000378:metabolism; D004257:DNA Polymerase II / Q000378:metabolism; D007700:Kinetics; D007942:Leukemia, Experimental / Q000201:enzymology; D008324:Mammary Tumor Virus, Mouse / Q000201:enzymology*; D051379:Mice; D012194:RNA-Directed DNA Polymerase / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases / Q000302:isolation & purification / Q000378:metabolism; D013698:Templates, Genetic
D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D004257:DNA Polymerase II; D012260:Ribonucleases