Analysis of the ribonuclease H activity of HIV-1 reverse transcriptase using RNA.DNA hybrid substrates derived from the gag region of HIV-1.
The RNase H activity associated with recombinant p66/p51 HIV-1 reverse transcriptase (RT) has been analyzed in the absence of DNA synthesis by using homogeneous RNA.DNA substrates. The substrates consisted of SP6 runoff transcripts from a portion of the gag region of the HIV-1 genome hybridized to complementary single-stranded DNA from either an M13 subclone or a phagemid transcription vector subclone. The corresponding hybrids either carried a 5'-mismatch of seven nucleotides or were fully base-paired. Analysis of recombinant HIV-1 p66/p51 RT by an activated gel assay employing these substrates suggested that the RNase H activity was exclusively associated with the p66 polypeptide. Denaturing gel electrophoresis was used to analyze the oligonucleotide products generated by hydrolysis of the hybrids by HIV-1 RT, M-MuLV RT, and Escherichia coli RNase H. The significant difference in the time-dependent distribution of products of HIV-1 RT vs E. coli RNase H catalyzed cleavage of 5'-mismatched hybrids indicated that the preparation of recombinant HIV-1 RT was free of contaminating bacterial RNase H. Although the HIV-1 RT associated RNase H activity shares many of the general mechanistic features of other retroviral enzymes [Gerard, G. F. (1981) Biochemistry 20, 256-265], the appearance of unique intermediates and end products in the course of hydrolysis of 5'-mismatched and fully base-paired hybrids indicated a significant difference in the sequence dependence of the kinetics of RNase H cleavage by HIV-1 RT and M-MuLV RT.
Mizrahi
V
V
Department of Macromolecular Sciences, Smith Kline and French Research and Development Laboratory, King of Prussia, Pennsylvania 19406.
D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004722:Endoribonucleases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D015750:Genes, gag; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D009693:Nucleic Acid Hybridization; D012367:RNA, Viral / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D016914:Ribonuclease H; D013379:Substrate Specificity
D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H