Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids.
The mode of action of the RNase H activity from HIV-1 was analyzed with a purified recombinant p66/p51 reverse transcriptase RT/RNase H protein and RNA-DNA hybrid consisting of RNA harboring the polypurine tract (ppt) and three complementary synthetic DNA oligonucleotides. Upon incubation of this preformed RNA-DNA hybrid with the p66/p51 RT/RNase H, a cleavage pattern is observed that indicates endonucleolytic RNase H activity with some sequence specificity for the next to last nucleotide of the 3'-end of the ppt RNA and one cut within the ppt. The RNase H avoids cleavage of G or A stretches. During RNA-directed DNA synthesis the RNA is hydrolyzed in a concerted action of RT and RNase H whereby the RNase H exhibits endonuclease as well as 3'-5'-exonuclease activity. The distance between the active centers of the RT and RNase H corresponds to 18 base pairs of the RNA-DNA hybrid. Plus-strand DNA-directed DNA synthesis initiates exactly at the next to last nucleotide of the 3'-end of the ppt RNA by means of the RNase H activity.
Wöhrl
B M
BM
Max-Planck-Institut fuer Molekulare Genetik, Abteilung Schuster, Berlin, FRG.
Moelling
K
K
D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D004279:DNA, Viral / Q000096:biosynthesis; D004722:Endoribonucleases / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H; D013379:Substrate Specificity
D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D009838:Oligodeoxyribonucleotides; D012367:RNA, Viral; D004247:DNA; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H