Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.26.4 (RNase H)
2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Alanine scanning mutagenesis was undertaken to evaluate the structural significance of Met230-His235 of the 66 kDa subunit of p66/p51 human immunodeficiency virus reverse transcriptase (HIV-1 RT). Together with Glu224-Trp229, these residues provide the framework of the p66 "primer grip", whose proposed role is maintaining the primer terminus in an orientation appropriate for nucleophilic attack on an incoming dNTP. Of these residues, altering Leu234 results in a p66 subunit incapable of associating into heterodimer. The remaining selectively mutated enzymes were successfully reconstituted and purified to homogeneity for evaluation of RT-associated activities. We show here that alterations to any residue within the p66-Trp229-Met230-Gly231-Tyr232-quartet alter functions associated with both the DNA polymerase and ribonuclease H (RNase H) domains. Detailed analysis of mutant p66Y232A/p51 with an intact or a model "precleaved" RNA-DNA hybrid suggests an altered RNase H phenotype could result from relocation of template-primer in the nucleic acid binding cleft. As a consequence, template nucleotide-8 is positioned in the immediate vicinity of the RNase H catalytic center rather than nucleotide-17.
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PMID:Alterations to the primer grip of p66 HIV-1 reverse transcriptase and their consequences for template-primer utilization. 867 16

The Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus and a causative agent of the Acquired Immuno Deficiency Syndrome (AIDS). Retroviruses are distinct from other viruses in their ability to encode an enzyme called reverse transcriptase (RT). The RT is the enzyme mainly involved in replication. It performs RNA- as well as DNA-dependent DNA synthesis in order to convert the single-stranded viral RNA genome into double-stranded DNA. The double-stranded DNA is stably integrated into the host cell genome and is used as a template for the production of a new viral generation. The HIV-1 RT is partially encoded by the POL open reading frame of the HIV-1 genome and consists of two subunits of 66 kDa (p66) and 51 kDa (p51). The p66 polypeptide encodes the reverse transcriptase and the RNase H domain. Half of the p66 molecules are further processed to generate the p51 protein with an identical N-terminus, but lacking the C-terminus which encodes the RNase H domain. In vivo both polypeptides are found in equimolar amounts thus forming a heterodimer. This dimerization is critical for the enzymatic activity. In this review we summarize (i) the replication cycle of HIV-1, (ii) the enzymatic properties of HIV-1 RT and (iii) the structure-function relationship of the HIV-1 RT in view of the known three dimensional structure.
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PMID:Human Immunodeficiency Virus type 1 reverse transcriptase. 886 66

Alterations to the highly conserved Asp549 of the retroviral ribonuclease H (RNase H) domain were evaluated in the heterodimeric (p66/p51) reverse transcriptases of human immunodeficiency and equine infectious anemia viruses. In addition to the polymerization-dependent and -independent modes of template hydrolysis, mutants were evaluated via their ability to select and extend the 3' polypurine tract (PPT) primers of these two lentiviruses into (+) strand DNA. Concerted and two-step reactions were designed to evaluate (+) strand priming, the latter of which allows discrimination between selection end extension events. In contrast to enzyme mutated at the highly conserved Glu478, substitution of Asp549 with Asn or Ala reduces, rather than completely eliminates, RNase H activity. When the requirement for RNase H function becomes more stringent, differences in activity are readily evident, most notably in the cleavage events liberating the 5' terminus of the PPT primer. PPT selection thus appears to represent a specialized form of RNase H activity that is more sensitive to minor structural alterations within this domain and may provide a novel therapeutic target.
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PMID:Substituting a conserved residue of the ribonuclease H domain alters substrate hydrolysis by retroviral reverse transcriptase. 907 91

In order to investigate how primer grip residues of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) contribute toward the architecture of its palm subdomain and neighboring structural elements, the DNA polymerase and ribonuclease H (RNase H) activities of enzymes bearing aromatic substitutions at Trp229 and Tyr232 of the catalytically-competent p66 subunit were evaluated. Although all mutants retained RNase H function, the manner in which different RNA-DNA hybrids were hydrolyzed was affected. Depending on the nature of the substitution, DNA-dependent DNA synthesis was (i) unaffected, (ii) interrupted shortly after initiation, or (iii) stalled when the replication machinery encountered an intramolecular duplex on the single-stranded template. Evaluating (-) strand strong-stop DNA synthesis on an RNA template derived from the viral genome raises the additional possibility that DNA and RNA primers might be differentially recognized by the retroviral polymerase. In support of this, all mutants were unable to extend the HIV-1 polypurine tract (PPT) RNA primer into (+) strand DNA, despite supporting the equivalent event from an oligodeoxynucleotide primer. Collectively, our data illustrate that subtle alterations to primer grip architecture may manifest themselves in discrimination between oligoribo- and oligodeoxyribonucleic acid primers.
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PMID:Mutating a conserved motif of the HIV-1 reverse transcriptase palm subdomain alters primer utilization. 915 16

Treatment of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with N-ethylmaleimide (NEM) selectively inhibits the RNase H activity. The cysteine residue at position 280 (C280) is the target for NEM; HIV-1 RT carrying the mutation C280S is resistant to NEM. Since HIV-1 RT is composed of two related subunits (p66 and p51) that play distinct roles, we asked whether the C280 in p51 or the C280 in p66 is responsible for the sensitivity of the enzyme to NEM. HIV-1 RT versions were prepared that had one mutant and one wild-type subunit. When these chimeric enzymes were tested, both the p51 and p66 subunits were found to contribute to the sensitivity of the enzyme to NEM. The implications of these results are discussed in the context of the structure of the enzyme.
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PMID:Subunit-specific mutagenesis of the cysteine 280 residue of the reverse transcriptase of human immunodeficiency virus type 1: effects on sensitivity to a specific inhibitor of the RNase H activity. 918 46

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.
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PMID:Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein. 919 28

The conformation of the DNA and the interactions of the nucleic acid with the protein in a complex of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and 19-mer/18-mer double-stranded DNA template-primer (dsDNA) are described. The structure of this HIV-1 RT complex with dsDNA serves as a useful paradigm for studying aspects of nucleotide polymerases such as catalysis, fidelity, drug inhibition, and drug resistance. The bound dsDNA has a bend of approximately 41 degrees at the junction of an A-form region (first five base pairs near the polymerase active site) and a B-form region (the last nine base pairs toward the RNase H active site). The 41 degrees bend occurs smoothly over the four base pairs between the A-form portion and the B-form portion in the vicinity of helices alpha H and alpha I of the p66 thumb subdomain. The interactions between the dsDNA and protein primarily involve the sugar-phosphate backbone of the nucleic acid and structural elements of the palm, thumb, and RNase H of p66, and are not sequence specific. Amino acid residues from the polymerase active site region, including amino acid residues of the conserved Tyr-Met-Asp-Asp (YMDD) motif and the "primer grip," interact with 3'-terminal nucleotides of the primer strand and are involved in positioning the primer terminal nucleotide and its 3'-OH group at the polymerase active site. Amino acid residues of the "template grip" have close contacts with the template strand and aid in positioning the template strand near the polymerase active site. Helix alpha H of the p66 thumb is partly inserted into the minor groove of the dsDNA and helix alpha I is directly adjacent to the backbone of the template strand. Amino acid residues of beta 1', alpha A', alpha B', and the loop containing His539 of the RNase H domain interact with the primer strand of the dsDNA.
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PMID:Protein-nucleic acid interactions and DNA conformation in a complex of human immunodeficiency virus type 1 reverse transcriptase with a double-stranded DNA template-primer. 935 57

Recently, tRNALys-3 was cross-linked via its anticodon loop to human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) between residues 230 and 357 (Mishima, Y., and Steitz, J. A. (1995) EMBO J. 14, 2679-2687). Scanning the surface of this region identified three basic amino acids Lys249, Arg307, and Lys311 flanking a small crevice on the p66 thumb subdomain outside the primer-template binding cleft. To assess an interaction of this region with the tRNA anticodon loop, these p66 residues were altered to Glu or Gln. p66 subunits containing K249Q, K311Q, K311E, and a dual R307E/K311E mutation formed a stable dimer with wild type p51. All mutants showed reduced affinity for tRNALys-3 and supported significantly less (-)-strand DNA synthesis from this primer than the parental heterodimer. In contrast, these variants efficiently synthesized HIV-1 (-)-strand strong-stop DNA from oligonucleotide primers and had minimal effect on RNase H activity, retaining endonucleolytic and directed cleavage of an RNA/DNA hybrid. Structural features of binary RT.tRNALys-3 complexes were examined by in situ footprinting, via susceptibility to 1, 10-phenanthroline-copper-mediated cleavage. Unlike wild type RT, mutants p66(K311Q)/p51 and p66(K311E)/p51 failed to protect the tRNA anticodon domain from chemical cleavage, indicating a significant structural alteration in the binary RT.tRNA complex. These results suggest a crevice in the p66 thumb subdomain of HIV-1 RT supports an interaction with the tRNALys-3 anticodon loop critical for efficient (-)-strand DNA synthesis.
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PMID:Mutating a region of HIV-1 reverse transcriptase implicated in tRNA(Lys-3) binding and the consequences for (-)-strand DNA synthesis. 960 66

The reverse transcriptase (RT) of HIV-1 and feline immunodeficiency virus (FIV) consist of two subunits of 51 kDa (p51) and 66 kDa (p66). In order to elucidate the role of p51 in the heterodimer, chimeric HIV-1/FIV RT heterodimers were constructed and characterized. The FIV RT p51/HIV-1 RT p66 chimera showed a 2.5-fold higher RNase H activity than the natural HIV-1 RT, a 50% lower strand displacement DNA synthesis activity and resistance to the two RT inhibitors 3'-azido-3'-deoxythymidine triphosphate (AZTTP) and Nevirapine. The HIV-1 RT p51/FIV RT p66 chimera on the other hand had very similar properties to the natural FIV RT. The differences observed upon exchange of the p51 subunits suggest that the three-dimensional structure of the p51 subunit in the RT heterodimers is not completely conserved between the human and the feline lentiviruses. Finally, our data suggest an important role for the p51 subunit in maintaining the optimal structural integrity of the RT heterodimer. The different effects of the small subunits on the sensitivity to known RT inhibitors might be of importance in the development of novel drugs against HIV-1 RT.
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PMID:Chimeric HIV-1 and feline immunodeficiency virus reverse transcriptases: critical role of the p51 subunit in the structural integrity of heterodimeric lentiviral DNA polymerases. 961 40

Small RNA pseudoknots, selected to bind human immunodeficiency virus type 1 (HIV-1) reverse transcriptase tightly, are potent inhibitors of reverse transcriptase. The co-crystal structure of reverse transcriptase complexed with a 33 nucleotide RNA pseudoknot has been determined by fitting the ligand into a high quality, 4-fold averaged 4.8 A resolution electron density map. The RNA is kinked between stems S1 and S2, thereby optimizing its contacts with subunits of the heterodimer. Its binding site extends along the cleft that lies between the polymerase and RNase H active sites, partially overlaps with that observed for duplex DNA and presumably overlaps some portion of the tRNA site. Stem S2 and loop L1 stabilize the 'closed' conformation of the polymerase through extensive electrostatic interactions with several basic residues in helix I of the p66 thumb and in the p66 fingers domain. Presumably, this RNA ligand inhibits reverse transcriptase by binding to a site that partly overlaps the primer-template binding site.
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PMID:The structure of HIV-1 reverse transcriptase complexed with an RNA pseudoknot inhibitor. 968 19


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