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

Ding, Jianping; Hughes, Stephen H.; Arnold, Edward

doi:10.1002/(sici)1097-0282(1997)44:2<125::aid-bip2>3.0.co;2-x

Cited by 57 publications

(11 citation statements)

References 65 publications

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“…molecular chimeric RT p51 subunits between HIV-1 and FIV RT enzymes were constructed and functionally tested. On the basis of the crystallized structures of HIV-1 RT (18,20,43,44) and the primary amino acid sequences of HIV-1 and FIV RT p51 subunits, three defined regions in HIV-1 RT p51 were replaced with the corresponding regions from FIV RT p51. The chimeric p51 subunits when reconstituted with HIV-1 RT p66 resulted in functional RT heterodimers ( Table 1).…”

Section: Discussionmentioning

confidence: 99%

“…In C2, the replaced region extends from the N terminus to the 6 loop and in C3 it extends from the 21 loop to the C terminus of HIV-1 RT p51. The choice of these regions was based on the current models of the crystallized structure of the HIV-1 RT heterodimer (18,20,43,44). Each of the individual chimeric HIV-1 or FIV RT p51 subunits was cloned with a hexahistidine tag at the 5′-end of the gene.…”

Section: Construction Of Intramolecular Chimeric Hiv-1-fiv Rtmentioning

confidence: 99%

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Intramolecular Chimeras of the p51 Subunit between HIV-1 and FIV Reverse Transcriptases Suggest a Stabilizing Function for the p66 Subunit in the Heterodimeric Enzyme

1999

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The human immunodeficiency virus (HIV) reverse transcriptase (RT) is a heterodimeric enzyme composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. Recently we showed that p51 plays an important role in the conformation of p66 within the HIV-1 RT heterodimer and hence appears to influence its catalytic activities [Amacker, M., and H ubscher, U. (1998) J. Mol. Biol. 278, 757-765]. This was further investigated here via construction of three intramolecular chimeras of HIV-1 and FIV RTs. The first 25 and 112 amino acids of the N terminus, respectively, as well as the last 22 amino acids of the C terminus in the p51 subunit of HIV-1 RT were exchanged with the corresponding regions of the FIV RT and combined with the wild-type HIV-1 p66. Characterization of these chimeric RT heterodimers demonstrated significant biochemical differences in (i) DNA-dependent DNA synthesis, (ii) strand displacement DNA synthesis, and (iii) RNase H activity. Our results indicate that both the N and C termini of HIV-1 RT p51 appear to be important in stabilizing the RT heterodimer for enzymatic functions.

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Section: Discussionmentioning

confidence: 99%

Section: Construction Of Intramolecular Chimeric Hiv-1-fiv Rtmentioning

confidence: 99%

Intramolecular Chimeras of the p51 Subunit between HIV-1 and FIV Reverse Transcriptases Suggest a Stabilizing Function for the p66 Subunit in the Heterodimeric Enzyme

1999

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“…3). In most crystal structures, the 3¢ end of the primer is located in the so-called P (priming) site [5,155]. In that configuration, the next incoming nucleotide can bind to the N (nucleotide-binding) site [104], and translocation to the P site occurs after the chemical step of dNTP incorporation, probably after PPi release [154].…”

Section: Effect Of the Next Complementary Nucleotide And Formation Ofmentioning

confidence: 99%

Nucleoside and nucleotide inhibitors of HIV-1 replication

Vivet-Boudou

Didierjean

Isel

et al. 2006

Cell. Mol. Life Sci.

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HIV-1 reverse transcriptase (RT) is one of the main targets for antiviral therapy. Two classes of RT inhibitors can be distinguished: those that are nucleoside or nucleotide analogues (sharing the common NRTIs abbreviation) and those that are not. This review focuses on the NRTIs, which are highly efficient in slowing down viral replication and are used in combination regimens. Unfortunately, the current inhibitors do not completely suppress viral replication and due to the high capacity of adaptation of HIV, allow the selection of drug-resistant viruses. Resistance mechanisms to NRTIs have been extensively investigated and can be divided into two types: improved discrimination of a nucleotide analogue relative to the natural substrate or increased phosphorolytic cleavage of an analogue-blocked primer. This knowledge is important both for the development of new drugs designed to target resistant strains and for the development of new antiviral strategies. The NRTIs currently in clinical trials and new developments in this area are also reviewed.

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“…The conformation of the nascent DNA duplexes observed in five DNA-polymerase-DNA complexes, HIV-1 reverse transcriptase (HIV-RT) (Ding et al, 1997;, Bacillus stearothermophilus polymerase I large fragment (B. stear) (Kiefer et al, (1998), the closed form of the Thermus aquaticus DNA polymerase I large fragment (KlenTaq) (Li et al, 1998), bacteriophage T7 DNApolymerase (T7) (Doublié et al, 1998) and rat DNA polymerase β-DNA-ddCTP (polβ) (Pelletier et al, 1994) whose coordinates have been deposited in the nucleic acids data base (Berman et al, 1992) are compared in Table II. The high negative values of Xdisplacement (X-disp), the positive inclination as well as the RMS values with the corresponding canonical DNA forms indicate that, the DNA duplexes bound to HIV-RT, B. stear, KlenTaq and T7 belong to the A-DNA family (Table II, Figure 2b).…”

Section: The Conformation Of the Template-primer Duplexes At The Vicimentioning

confidence: 99%

DNA Structure and Polymerase Fidelity: A New Role for A-DNA

Timsit

2000

Journal of Biomolecular Structure and Dynamics

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Although the recent structural studies on polymerases have brought new insights on polymerase fidelity, the role of DNA sequence and structure is not well understood. Here, the analysis of the crystal structures of hotspots for polymerase slippage shows that, in the B- form, these sequences share common structural alterations which may explain the high rate of replication errors. In (CA)(n) tracts, a "Janus-like" structure with shifted base pairs in the major groove but an apparent normal geometry in the minor groove constitutes a molecular decoy which can mislead the polymerases. A model of the rat polymerase β bound to this structure suggests that an altered conformation of the nascent template-primer duplex can interfere with correct nucleotide incorporation by affecting the geometry of the active site and breaking the rules of base pairing while at the same time escaping enzymatic mechanisms of error discrimination scanning for the correct geometry of the minor groove. In contrast, by showing that the A-form greatly attenuates the sequence-dependent structural alterations in hotspots, this study reveals that the A-conformation of the nascent template-primer duplex at the vicinity of the polymerase active site will contribute to fidelity. The A-form may play the role of a structural buffer which preserves the correct geometry of the active site for all sequences. The comparison of the conformation of the nascent template-primer duplex in five available crystal structures of DNA polymerase-DNA complexes shows indeed that polymerase β the least accurate enzyme, is unique in binding to a B-DNA duplex even close to its active site. This model leads to several predictions which are discussed in the light of published experimental data.

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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

Cited by 57 publications

References 65 publications

Intramolecular Chimeras of the p51 Subunit between HIV-1 and FIV Reverse Transcriptases Suggest a Stabilizing Function for the p66 Subunit in the Heterodimeric Enzyme

Intramolecular Chimeras of the p51 Subunit between HIV-1 and FIV Reverse Transcriptases Suggest a Stabilizing Function for the p66 Subunit in the Heterodimeric Enzyme

Nucleoside and nucleotide inhibitors of HIV-1 replication

DNA Structure and Polymerase Fidelity: A New Role for A-DNA

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