Vaccinia virus polyriboadenylate polymerase: convalent linkage of the product with polyribonucleotide and polydeoxyribonucleotide primers.
A POLYRIBOADENYLATE [POLY(A)] POLYMERASE, PURIFIED FROM VACCINIA VIRUS CORES, WAS STIMULATED BY POLYDEOXYRIBOADENYLATE: polydeoxyribothymidylate [poly(dA:dT)] and by polyribocytidylate [poly(C)] primers suggesting mechanisms of either transcription or terminal addition. Evidence for the latter was obtained by the demonstration of covalent linkages between the poly(A) products and both primers. In 99% dimethylsulfoxide-sucrose gradients, the sedimentation of poly(A) formed with poly(dA: dT) primer was reduced after DNase I treatment and the sedimentation of poly(A) formed with poly(C) primer was reduced by RNase A treatment, whereas the sedimentation of poly(A) formed without primer was not affected by either. Formation of a phosphodiester bond between primer and product was demonstrated by means of isotope transfer experiments. (32)P from alpha-[(32)P]ATP was transferred to 2'(3')-CMP after alkaline or enzymatic hydrolysis of the poly(C)-primed polymerase reaction product. Transfer primarily or exclusively to 3'-dTMP was found after enzymatic hydrolysis of the poly(dA: dT)-primed polymerase reaction product. The elution pattern of the poly(A) polymerase from DNA-cellulose suggested that a single enzyme catalyzes the attachment of adenylate residues to both polyribonucleotide and polydeoxyribonucleotide primers; nevertheless the purest enzyme preparations contain two bands resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulfate.
D000255:Adenosine Triphosphate; D001345:Autoradiography; D002501:Centrifugation, Zonal; D002845:Chromatography; D003851:Deoxyribonucleases; D004121:Dimethyl Sulfoxide; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D009841:Oligonucleotides; D012260:Ribonucleases; D013268:Stimulation, Chemical; D014616:Vaccinia virus / Q000201:enzymology*
D009841:Oligonucleotides; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D003851:Deoxyribonucleases; D012260:Ribonucleases; D004121:Dimethyl Sulfoxide