Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein

The human immunodeficiency virus type 1 nucleocapsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and thermodynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quadruplexes formed by the sequence d(GGTTGGTGTGGTTGG) in the presence of K+ or Sr2+. Circular dichroism studies demonstrate that NC effectively unfolds the quadruplexes. Studies carried out with NC variants suggest that destabilization is mediated by the zinc fingers of NC. Calorimetric studies reveal that NC destabilization is enthalpic in origin, probably owing to unstacking of the G-quartets upon protein binding. In contrast, parallel studies performed on a related DNA duplex reveal that under conditions where NC readily destabilizes and unfolds the quadruplexes, its effect on the DNA duplex is much less pronounced. The differences in NC's ability to destabilize quadruplex versus duplex is in accordance with the higher ΔG of melting for the latter, and with the inverse correlation between nucleic acid stability and the destabilizing activity of NC.

[1]  B. Roques,et al.  Role of the structure of the top half of HIV-1 cTAR DNA on the nucleic acid destabilizing activity of the nucleocapsid protein NCp7. , 2004, Journal of molecular biology.

[2]  F. Chen,et al.  Sr2+ facilitates intermolecular G-quadruplex formation of telomeric sequences. , 1992, Biochemistry.

[3]  M. Summers,et al.  Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus , 2004, Nature.

[4]  B. Kankia,et al.  Optical absorption assay for strand-exchange reactions in unlabeled nucleic acids. , 2004, Nucleic acids research.

[5]  N Bischofberger,et al.  The tertiary structure of a DNA aptamer which binds to and inhibits thrombin determines activity. , 1993, Biochemistry.

[6]  B. Kankia.  Hydration effects of Ni(2+) binding to synthetic polynucleotides with regularly alternating purine-pyrimidine sequences. , 2000, Nucleic acids research.

[7]  K. Musier-Forsyth,et al.  Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism. , 2005, Progress in nucleic acid research and molecular biology.

[8]  N. Jing,et al.  Structure-Activity of Tetrad-forming Oligonucleotides as a Potent Anti-HIV Therapeutic Drug* , 1998, The Journal of Biological Chemistry.

[9]  V. Marathias,et al.  Structures of the potassium-saturated, 2:1, and intermediate, 1:1, forms of a quadruplex DNA. , 2000, Nucleic acids research.

[10]  P. Barbara,et al.  Secondary structure and secondary structure dynamics of DNA hairpins complexed with HIV-1 NC protein. , 2004, Biophysical journal.

[11]  K. Breslauer,et al.  Enthalpy-entropy compensations in drug-DNA binding studies. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[12]  B. Roques,et al.  HIV-1 nucleocapsid protein activates transient melting of least stable parts of the secondary structure of TAR and its complementary sequence. , 2002, Journal of Molecular Biology.

[13]  J. Berg,et al.  Potential metal-binding domains in nucleic acid binding proteins. , 1986, Science.

[14]  L. Marky,et al.  Folding of the thrombin aptamer into a G-quadruplex with Sr(2+): stability, heat, and hydration. , 2001, Journal of the American Chemical Society.

[15]  T. Copeland,et al.  Primary structure of the low molecular weight nucleic acid-binding proteins of murine leukemia viruses. , 1981, The Journal of biological chemistry.

[16]  P. Charneau,et al.  A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication , 1992, Journal of virology.

[17]  Y. Pommier,et al.  Ion selective folding of loop domains in a potent anti-HIV oligonucleotide. , 1997, Biochemistry.

[18]  B. Roques,et al.  Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein. , 1997, Biopolymers.

[19]  R. Lumry,et al.  Enthalpy–entropy compensation phenomena in water solutions of proteins and small molecules: A ubiquitous properly of water , 1970, Biopolymers.

[20]  J. Bess,et al.  Molecular Requirements for Human Immunodeficiency Virus Type 1 Plus-Strand Transfer: Analysis in Reconstituted and Endogenous Reverse Transcription Systems , 1999, Journal of Virology.

[21]  J. Casas-Finet,et al.  Sequence-Specific Binding of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein to Short Oligonucleotides , 1998, Journal of Virology.

[22]  Jianhui Guo,et al.  Zinc Finger Structures in the Human Immunodeficiency Virus Type 1 Nucleocapsid Protein Facilitate Efficient Minus- and Plus-Strand Transfer , 2000, Journal of Virology.

[23]  H. Buc,et al.  HIV-1 reverse transcription. A termination step at the center of the genome. , 1994, Journal of molecular biology.

[24]  B. Roques,et al.  Nucleic acid sequence discrimination by the HIV-1 nucleocapsid protein NCp7: a fluorescence study. , 1999, Biochemistry.

[25]  K. Musier-Forsyth,et al.  Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Jean-Louis Mergny,et al.  Following G‐quartet formation by UV‐spectroscopy , 1998, FEBS letters.

[27]  J. Feigon,et al.  Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[28]  K. Breslauer,et al.  Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves , 1987, Biopolymers.

[29]  B. Grinde,et al.  Mutations in the central polypurine tract of HIV-1 result in delayed replication. , 1992, Virology.

[30]  S. L. Le Grice,et al.  Role of Post-transcriptional Modifications of Primer tRNALys,3 in the Fidelity and Efficacy of Plus Strand DNA Transfer during HIV-1 Reverse Transcription* , 1999, The Journal of Biological Chemistry.

[31]  Ronald M. Levy,et al.  Entropy−Enthalpy Compensation in Solvation and Ligand Binding Revisited , 1998 .

[32]  K. Musier-Forsyth,et al.  Specific zinc-finger architecture required for HIV-1 nucleocapsid protein's nucleic acid chaperone function , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[33]  J. Darlix,et al.  The chaperoning and assistance roles of the HIV-1 nucleocapsid protein in proviral DNA synthesis and maintenance. , 2004, Current HIV research.

[34]  B. Roques,et al.  Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7. , 2003, Journal of molecular biology.

[35]  S. Covey.  Amino acid sequence homology in gag region of reverse transcribing elements and the coat protein gene of cauliflower mosaic virus. , 1986, Nucleic acids research.

[36]  J G Levin,et al.  Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA , 1997, Journal of virology.

[37]  E. Le Cam,et al.  Human Immunodeficiency Virus Type 1 Central DNA Flap: Dynamic Terminal Product of Plus-Strand Displacement DNA Synthesis Catalyzed by Reverse Transcriptase Assisted by Nucleocapsid Protein , 2001, Journal of Virology.

[38]  N. Jullian,et al.  Structural and dynamic characterization of the aromatic amino acids of the human immunodeficiency virus type I nucleocapsid protein zinc fingers and their involvement in heterologous tRNA(Phe) binding: a steady-state and time-resolved fluorescence study. , 1993, Biophysical journal.

[39]  R. Karpel,et al.  HIV-1 nucleocapsid protein as a nucleic acid chaperone: spectroscopic study of its helix-destabilizing properties, structural binding specificity, and annealing activity. , 2002, Journal of molecular biology.

[40]  Mary Lapadat-Tapolsky,et al.  Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1 , 1995, Nucleic Acids Res..

[41]  M. Summers,et al.  NMR structure of the HIV-1 nucleocapsid protein bound to stem-loop SL2 of the psi-RNA packaging signal. Implications for genome recognition. , 2000, Journal of molecular biology.

[42]  B. Roques,et al.  First glimpses at structure-function relationships of the nucleocapsid protein of retroviruses. , 1995, Journal of molecular biology.

[43]  R. Gorelick,et al.  Subtle Alterations of the Native Zinc Finger Structures Have Dramatic Effects on the Nucleic Acid Chaperone Activity of Human Immunodeficiency Virus Type 1 Nucleocapsid Protein , 2002, Journal of Virology.

[44]  J. DeStefano,et al.  Differing Roles of the N- and C-terminal Zinc Fingers in Human Immunodeficiency Virus Nucleocapsid Protein-enhanced Nucleic Acid Annealing* , 2003, Journal of Biological Chemistry.

[45]  W. Gmeiner,et al.  NMR Structure of the Thrombin-Binding DNA Aptamer Stabilized by Sr2+ , 2003, Journal of biomolecular structure & dynamics.

[46]  E. Vermaas,et al.  Selection of single-stranded DNA molecules that bind and inhibit human thrombin , 1992, Nature.

[47]  B. Roques,et al.  Binding of the HIV-1 Nucleocapsid Protein to the Primer tRNA, inVitro, Is Essentially Not Specific (*) , 1995, The Journal of Biological Chemistry.

[48]  Karin Musier-Forsyth,et al.  Mechanistic insights into the kinetics of HIV-1 nucleocapsid protein-facilitated tRNA annealing to the primer binding site. , 2004, Journal of molecular biology.

[49]  R. Gorelick,et al.  G-quartets direct assembly of HIV-1 nucleocapsid protein along single-stranded DNA. , 2003, Nucleic acids research.

[50]  R. Shafer,et al.  Lead is unusually effective in sequence-specific folding of DNA. , 2000, Journal of molecular biology.

[51]  E. Le Cam,et al.  G-quartets assembly within a G-rich DNA flap. A possible event at the center of the HIV-1 genome. , 2002, Nucleic acids research.

[52]  S. Balasubramanian,et al.  Selection of zinc fingers that bind single-stranded telomeric DNA in the G-quadruplex conformation. , 2001, Biochemistry.

[53]  J. Berg,et al.  Retroviral nucleocapsid protein-metal ion interactions: folding and sequence variants. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[54]  M. Rosbash,et al.  A high affinity binding site for the HIV-1 nucleocapsid protein. , 1997, Nucleic acids research.

[55]  B. Roques,et al.  Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers. , 1992, Proceedings of the National Academy of Sciences of the United States of America.