RNase H activity associated with bacterially expressed reverse transcriptase of human T-cell lymphotropic virus III/lymphadenopathy-associated virus.

We have investigated the binding of human immunodeficiency virus reverse transcriptase (HIV-RT) to various hybrid RNA-DNA or DNA-DNA nucleic acid structures. Binding was measured by preequilibrating the RT with the nucleic acid substrate in the presence or absence of Mg2+ and then initiating synthesis or RNase H degradation reactions in the presence of excess "trap" polymer [poly(rA)-oligo(dT)]. The trap polymer sequestered RT molecules as soon as they dissociated from the substrate, such that the amount of synthesis or degradation on the substrate was proportional to the amount of bound RT. On hybrid substrates that had the 3' terminus of a complementary DNA oligomer recessed on a longer DNA or RNA template, binding to the RNA-DNA hybrid was more stable. Both the dissociation rate constant (k(off)) and equilibrium constant (Kd) values were larger for the DNA-DNA substrates by 5-10-fold. The difference was clearly in dissociation, since the association rate constant (k(on)) for both types of substrates was similar. On hybrid structures that had the 3' termini of a complementary RNA or DNA oligomer recessed on a longer DNA template, k(off) values are approximately the same on either structure. Although binding of the RT to DNA-DNA hybrid structures did not require Mg2+, its presence during the preequilibration period greatly stabilized binding. An approximate 20-60-fold decrease in the k(off), depending on the substrate structure, was observed with Mg2+. Measurements on one particular DNA-DNA hybrid indicated that the k(on) decreased by approximately 2 orders of magnitude with Mg2+. The relevance of these results to HIV replication is discussed.

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confidence: 99%

Parameters that influence the binding of human immunodeficiency virus reverse transcriptase to nucleic acid structures

DeStefano

Bambara²,

Fay³

1993

“…Thep66subunit contains both the polymerase and the RNase H activities. The p51 subunit, derived from a carboxyl terminal truncation of the p66 subunit, contains the polymerase domain, but lacks the RNase H domain (Hansen et al, 1987;Hizi et al, 1988;LeGrice et al, 1991;Lowe et al, 1988). A bireactantbiproduct mechanism has been proposed for DNA synthesis catalyzed by RT(Majundarm etal., 1988).…”

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confidence: 99%

Identification of the nucleotide binding site of HIV-1 reverse transcriptase using dTTP as a photoaffinity label

Cheng

Merrill²,

Painter³

et al. 1993

We have utilized UV-induced cross-linking of [methyl-3H]dTTP to identify the nucleotide binding site on heterodimeric HIV-1 reverse transcriptase (RT). RT was derivatized by irradiating a solution containing [methyl-3H]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 microM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Under these reaction conditions, only the 66-kDa subunit of the 66-kDa/51-kDa RT heterodimer was labeled with dTTP. The [methyl-3H]dTTP-labeled RT was fragmented with trypsin and endoproteinase Asp-N, and peptides were purified on reversed phase HPLC. The peptide covalently linked to [methyl-3H]dTTP was subjected to amino acid sequence analysis. The sequencing data localized the nucleotide binding site of RT to Lys-73 in the vicinity of several mutation sites linked to antiviral drug resistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to understand the basis for the antiviral activity of nucleoside analogs such as AZT, ddI, and ddC. This information may also be useful for a more rationally based design of anti-HIV agents.

“…e enzyme responsible for replication of the human immunodeficiency virus (HIV)1 genome, reverse transcriptase (RT), is both a DNA polymerase and a RNase H (Hansen et al, 1987(Hansen et al, , 1988Becerra et al, 1990). The native enzyme is a heterodimer composed of a 66-and 51-kDa subunit (Di Marzo-Veronese et al, 1986; Lightfoote et al, 1986).…”

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confidence: 99%

“…The native enzyme is a heterodimer composed of a 66-and 51-kDa subunit (Di Marzo-Veronese et al, 1986; Lightfoote et al, 1986). The RNase H active site is, at least in part, in the carboxyl-terminal domain of the 66-kDa polypeptide (Hansen et al, 1987(Hansen et al, , 1988; Davies et al, 1991).…”

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confidence: 99%

Human immunodeficiency virus reverse transcriptase ribonuclease H: specificity of tRNALys3-primer excision

Furfine

Reardon²

1991

Two model substrates were prepared to examine the mechanism of tRNA-primer excision catalyzed by reverse transcriptase associated ribonuclease H (RT-RNase H). The first model substrate contained sequences from the HIV genome and was designed to be structurally similar to the DNA-extended tRNA created by initiation of minus-strand DNA synthesis during retroviral replication. The DNA-extended RNA was a template and was annealed to a DNA oligonucleotide that primed reverse transcription of the RNA in the template. The second model substrate was structurally similar the first substrate but contained sequences unrelated to the HIV viral genome. The RT-RNase H catalyzed excision of the RNA from the template of the two model substrates was examined. Human immunodeficiency virus (HIV) and Moloney murine leukemia virus RT-RNase H hydrolyzed the substrates to leave a single ribonucleotide 5'-phosphate at the 5'-terminus of the model DNA genome. In contrast, avian myeloblastosis virus RT-RNase H hydrolyzed the phosphodiester bond at the DNA-RNA junction. These hydrolytic specificities were not highly dependent on substrate sequence. The importance of these specificities to retroviral integration is discussed. Additional data indicated that the HIV polymerase and RNase H active sites are separated by a distance equivalent to the length of a 15-nucleotide RNA-DNA heteroduplex.