Point mutations in conserved amino acid residues within the C‐terminal domain of HIV‐1 reverse transcriptase specifically repress RNase H function

Schatz, O.; Cromme, F. V.; Grüninger-Leitch, Fiona; Grice, Stuart F. J. Le

doi:10.1016/0014-5793(89)81559-5

Cited by 168 publications

(156 citation statements)

References 20 publications

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“…These were generated by incubating RNA transcripts containing 20-nt DNA primers at their 3Ј termini with HIV-1 p66E478Q/p51 (an RT devoid of RNase H activity) (24,25) and 200 M dNTPs in a buffer of 10 mM Tris-HCl, pH 8.0, 6 mM MgCl 2 , 80 mM NaCl, and 5 mM dithiothreitol for 60 min at 37°C. DNA synthesis was terminated by extraction with an equal volume of 1:1 phenol:chloroform (v/v), and the products precipitated in three volumes of ethanol, 0.1 volume of 3 M NaOAc, pH 5, and resuspended in nondenaturing gel-loading buffer (30% glycerol, 0.25% bromphenol blue, and 0.25% xylene cyanol).…”

Section: Construction and Purification Of Eiav And Hiv Rt Mutants-mentioning

confidence: 99%

“…3D indicate that although the highly conserved Asp 549 is clearly important for RNase H function, it is not absolutely critical, because low-level hydrolysis could still be achieved with both p66D549A/p51 mutants. Although slightly prolonged autoradiographic exposure was necessary to highlight the hydrolysis products from each enzyme, retention of function can be contrasted with an HIV-1 RT mutant containing the substitution Glu478Gln, which completely abolishes Mg 2ϩ -dependent RNase H activity (24,25). A second feature of both Asp 549 3 Ala mutants is again relaxed specificity of endonucleolytic cleavage.…”

Section: Dna Polymerase Activities Of Rnase H Mutants-p66/p51mentioning

confidence: 99%

“…5A takes advantage of the HIV-1 RNase H mutant p66E478Q/p51 (24,25), which is completely devoid of Mg 2ϩ -dependent RNase H activity (Fig. 3D) but retains full DNA polymerase activity.…”

Section: Two-step Reactions Delineating Ppt Selection and Extension-ementioning

confidence: 99%

“…W, wild-type p66/p51; N, HIV-1 p66D549N/p51; A, HIV-1 p66D549A/p51; EQ, HIV-1 p66E478Q/ p51. HIV-1 p66E478Q/p51 RT is devoid of Mg 2ϩ -dependent RNase H activity (24,36) and is used to extend primers selected by other enzymes while ensuring that no further RNA processing occurs during the "extension" portion of the assay. The HIV 3Ј PPT sequence is bracketed, and (ϩ) strand initiation sites are indicated with arrows.…”

Section: Two-step Reactions Delineating Ppt Selection and Extension-ementioning

confidence: 99%

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Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Rausch

Grice

1997

Journal of Biological Chemistry

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Alterations to the highly conserved Asp 549 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 Glu 478 , substitution of Asp 549 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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Section: Construction and Purification Of Eiav And Hiv Rt Mutants-mentioning

confidence: 99%

Section: Dna Polymerase Activities Of Rnase H Mutants-p66/p51mentioning

confidence: 99%

“…5A takes advantage of the HIV-1 RNase H mutant p66E478Q/p51 (24,25), which is completely devoid of Mg 2ϩ -dependent RNase H activity (Fig. 3D) but retains full DNA polymerase activity.…”

Section: Two-step Reactions Delineating Ppt Selection and Extension-ementioning

confidence: 99%

Section: Two-step Reactions Delineating Ppt Selection and Extension-ementioning

confidence: 99%

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Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Rausch

Grice

1997

Journal of Biological Chemistry

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“…A RNase H domain is also present at the C-terminus of retroviral reverse transcriptase which converts a single-stranded retroviral genomic RNA into a double-stranded DNA for integration into host chromosomes, thus playing a significant role in the HIV reverse transcription process [3]. In vivo studies demonstrated that inactivation of RNase H results in non-infectious virus particles [4,5].…”

Section: Introductionmentioning

confidence: 99%

Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

Elsässer

Fels

2010

J Mol Model

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International audienceRibonuclease enzymes (RNases) play key roles in the maturation and metabolism of all RNA molecules. Computational simulations of the processes involved can help to elucidate the underlying enzymatic mechanism and is often employed in a synergistic approach together with biochemical experiments. Theoretical calculations require atomistic details regarding the starting geometries of the molecules involved, which, in the absence of crystallographic data, can only be achieved from computational docking studies. Fortunately, docking algorithms have improved tremendously in recent years, so that reliable structures of enzyme-ligand complexes can now be successfully obtained from computation. However, most docking programs are not particularly optimized for nucleotide docking. In order to assist our studies on the cleavage of RNA by the two most important ribonuclease enzymes, RNase A and RNase H, we evaluated four docking tools--MOE2009, Glide 5.5, QXP-Flo+0802, and Autodock 4.0--for their ability to simulate complexes between these enzymes and RNA oligomers. To validate our results, we analyzed the docking results with respect to the known key interactions between the protein and the nucleotide. In addition, we compared the predicted complexes with X-ray structures of the mutated enzyme as well as with structures obtained from previous calculations. In this manner, we were able to prepare the desired reaction state complex so that it could be used as the starting structure for further DFT/B3LYP QM/MM reaction mechanism studies

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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

1997

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Point mutations in conserved amino acid residues within the C‐terminal domain of HIV‐1 reverse transcriptase specifically repress RNase H function

Cited by 168 publications

References 20 publications

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Substituting a Conserved Residue of the Ribonuclease H Domain Alters Substrate Hydrolysis by Retroviral Reverse Transcriptase

Nucleotide docking: prediction of reactant state complexes for ribonuclease enzymes

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

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