Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease.
The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.
Oyama
F
F
Mitsubishi Kasei Institute of Life Sciences, Tokyo, Japan.
Kikuchi
R
R
Crouch
R J
RJ
Uchida
T
T
D001354:Avian Leukosis Virus / Q000201:enzymology*; D009189:Avian Myeloblastosis Virus / Q000201:enzymology*; D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D009052:Leukemia Virus, Murine / Q000201:enzymology; D009693:Nucleic Acid Hybridization; D010047:Ovalbumin / Q000235:genetics; D012313:RNA / Q000378:metabolism; D000079962:RNA, Circular; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000502:physiology*
D003854:Deoxyribonucleotides; D000079962:RNA, Circular; D012333:RNA, Messenger; D012313:RNA; D010047:Ovalbumin; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H