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2001 | 48 | 2 | 511-523
Article title

Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate.

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Potassium permanganate oxidation of pyrimidine bases is often used to probe single-stranded regions in functional DNA-protein complexes. However, so far reactivity of these bases in double-stranded DNA has not been studied quantitatively. We have investigated the kinetics of oxidation of pyrimidines in supercoiled pDS3 plasmid dsDNA by quantitative KMnO4 footprinting, in connection with parallel studies on the effect of Mg2+ on kinetics of oxidation of individual thymines in the single-stranded region of the open transcription complex of Escherichia coli RNA polymerase at a cognate Pa promoter contained in this plasmid. Rate constants of oxidation for pyrimidines, kj, in selected regions of pDS3 DNA, including Pa promoter, were determined under single-hit reaction conditions in the absence and presence of 10 mM MgCl2. Their values appeared to be sequence-dependent and were: (i) the largest for Ts in 5'TA3' and 5'TC3' steps, while 2-4 times smaller for 5'-adjacent ones in TT(A,G,C) and TTT(A) runs, (ii) for Cs in 5'TC3' steps 2-4 fold smaller than for adjacent Ts, and (iii) in the presence of Mg2+ generally larger by a sequence-dependent factor: in 5'TC3' steps of about 2 and 4 for Ts and Cs, respectively, in 5'TA3' steps of TTA and TTTA sequences for 3'-terminal Ts of about 3, while for their 5'-neighbors of a distinctly smaller value of about 2. Comparison of kj data for corresponding Ts located between +1 and -10 regions of Pa promoter in dsDNA and in ssDNA form in the open transcription complex, reported elsewhere, demonstrates that reactivity of pyrimidines in dsDNA is by 2-3 orders of magnitude smaller. The effect of Mg2+ in dsDNA is interpreted in terms of electrostatic barrier to diffusion of MnO4- on DNA surface, which is lowered by diffusive binding of these ions to backbone phosphates, involving also sequence-specific contacts with bases in the minor and major grooves of B-DNA.
Physical description
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
  • Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland
  • 1. Hayatsu, H. & Ukita, T. (1967) The selective degradation of pyrimidines in nucleic acids by permanganate oxidation. Biochem. Biophys. Res. Commun. 29, 556-561.
  • 2. Iida, S. & Hayatsu, H. (1971) The permanganate oxidation of deoxyribonucleic acid. Biochim. Biophys. Acta 240, 370-375.
  • 3. McCarthy, J.G., Williams, L.D. & Rich, A. (1990) Chemical reactivity of potassium permanganate and diethyl pyrocarbonate with B DNA: Specific reactivity with short A-tracts. Biochemistry 29, 6071-6081.
  • 4. Jeppsen, C. & Nielsen, P.E. (1988) Detection of intercalation-induced changes in DNA structure by reaction with diethyl pyrocarbonate and potassium permanganate. FEBS Lett. 231, 172-176.
  • 5. Sasse-Dwight, S. & Gralla, J.D. (1991) Footprinting of protein-DNA complexes in vivo. Methods Enzymol. 208, 146-168.
  • 6. Nielsen, P.E., Egholm, M., Berg, R.H. & Buchardt, O. (1991) Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 254, 1497-1500.
  • 7. Nielsen, P.E., Egholm, M. & Buchardt, O. (1994) Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement. J. Mol. Recognit. 7, 165- 170.
  • 8. Sasse-Dwight, S. & Gralla, J.D. (1989) KMnO4 as a probe for lac promoter DNA melting and mechanism in vivo. J. Biol. Chem. 264, 8074-8081.
  • 9. Łoziński, T. & Wierzchowski, K.L. (2001) Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study. Acta Biochim. Polon. 48, 495-510.
  • 10. Łoziński, T., Markiewicz, W.T., Wyrzykiewicz, T.K. & Wierzchowski, K.L. (1989) Effect of the sequence-dependent structure of the 17 bp AT spacer on the strength of consensus-like E. coli promoters in vivo. Nucleic Acids Res. 17, 3855-3863.
  • 11. Łoziński, T., Adrych-Rożek, K., Markiewicz, W.T. & Wierzchowski, K.L. (1991) Effect of DNA bending in various regions of a consensus-like Escherichia coli promoter on its strength in vivo and structure of the open complex in vitro. Nucleic Acids Res. 19, 2947- 2953.
  • 12. Ide, H., Kow, Y.W. & Wallace, S.S. (1985) Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res. 13, 8035-8052.
  • 13. Tsodikov, O.V., Craig, M.L., Saecker, R.M. & Record, Jr., M.T. (1998) Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformation changes in protein- DNA complexes: Application to DNA opening by Eσ70 RNA polymerase. J. Mol. Biol. 283, 757-769.
  • 14. Mitas, M.Yu.A., Dill, J., Kamp, T.J., Chambers, E.J. & Haworth, I.S. (1995) Hairpin properties of single stranded DNA containing a GC-rich triplet repeat: (CTG)15. Nucleic Acids Res. 23, 1050-1059.
  • 15. Boutonnet, N., Hui, X. & Zakrzewska, K. (1993) Looking into the grooves of DNA. Biopolymers 33, 479-490.
  • 16. Gorin, A.A., Zhurkin, V.B. & Olson, W. (1995) B-DNA twisting correlates with base-pair morphology. J. Mol. Biol. 247, 34-48.
  • 17. Zakrzewska, K. (1992) Static and dynamic properties of AT sequences. J. Biomol. Struct. Dyn. 9, 681-693.
  • 18. Breslauer, K.J., Frank, R., Blöcker, H. & Marky, L.A. (1986) Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750.
  • 19. Leroy, J.L., Charretier, E., Kochoyan, M. & Gueron, M. (1988) Evidence from base-pair kinetics for two types of adenine tract structures in solution: Their relation to DNA structure. Biochemistry 27, 8894-8898.
  • 20. Patel, D.J. & Kozlowski, S.A. (1985) Conformation, dynamics, and structural transitions of the TATA box region of self-complementary d[(C-G)n- T-A-T-A-(C-G)n] duplexes in solution. Biochemistry 24, 926-935.
  • 21. Patel, D.J. & Kozlowski, S.A. (1985) Conformation and dynamics of the Pribnow box region of the self-complementary d(C-G-A-T-T- A-T-A-A-T-C-G) duplex in solution. Biochemistry 24, 936-944.
  • 22. Moe, J.G., Folta-Stogniew, E. & Russu, I.M. (1995) Energetics of base pair opening in a DNA dodecamer containing an A3T3 tract. Nucleic Acids Res. 23, 1984-1989.
  • 23. Lu, P., Cheung, S. & Arndt, K. (1983) Possible molecular determinants in the DNA structure of regulatory sequences. J. Biomol. Struct. Dyn. 1, 509-521.
  • 24. Ramstein, J. & Lavery, R. (1988) Energetic coupling between DNA bending and base pair opening. Proc. Natl. Acad. Sci. U.S.A. 85, 7231-7235.
  • 25. Misra, V.K. & Draper, D.E. (1999) The interpretation of Mg2+ binding isotherms for nucleic acids using Poisson-Boltzmann theory. J. Mol. Biol. 294, 1135-1147.
  • 26. Paulsen, M.D., Andersen, C.F. & Record, Jr., M.T. (1988) Counterion exchange reactions on DNA: Monte Carlo and Poisson-Boltzmann analysis. Biopolymers 27, 1249-1265.
  • 27. Manning, G.S. (1978) The molecular theory of polyelectrolyte solutions with applications to electrostatic properties of polynucleotides. Q. Rev. Biophys. 11, 179-246.
  • 28. Duguid, J., Bloomfield, V.A., Benevides, J. & Thomas, Jr., G.J. (1993) Raman spectroscopy of DNA-metal complexes. I. Interactions and conformational effects of the divalent cations: Mg, Ca, Sr, Ba, Mn, Co, Ni, Cu, Pd and Cd. Biophys J. 65, 1916-1928.
  • 29. Duguid, J., Bloomfield, V.A., Benevides, J. & Thomas, Jr., G.J. (1995) Raman spectroscopy of DNA-metal complexes: II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cd2+. Biophys J. 69, 2623-2641.
  • 30. Li, A.Z., Huang, H., Re, X., Qi, L.J. & Marx, K.A. (1998) A gel electrophoretic study of the competitive effects of monovalent counterion on the extent of divalent counterions binding to DNA. Biophys. J. 74, 964-973.
  • 31. Buckin, V.A., Kankiya, B.I., Rentzeperis, D. & Marky, L.A. (1994) Mg2+ recognizes the sequence of DNA through its hydration shell. J. Am. Chem. Soc. 116, 9423-9429.
  • 32. MacKerell, A.D. (1997) Influence of magnesium ions on duplex DNA structural, dynamic, and solvation properties. J. Phys. Chem. B, 101, 646-650.
  • 33. Brukner, I., Susic, S., Dlakic, M., Savic, A. & Pongor, S. (1994) Physiological concentration of magnesium ions induces a strong macroscopic curvature in GGGCCC-containing DNA. J. Mol. Biol. 236, 26-32.
  • 34. McFail-Isom, L., Shui, X. & Williams, L.D. (1998) Divalent cations stabilize unstacked conformations of DNA and RNA by interacting with base π systems. Biochemistry 37, 17105-17111.
  • 35. Sines, C.C., McFail-Isom, L., Howerton, S.B., VanDerveer, D. & Williams, L.D. (2000) Cations mediate B-DNA conformational heterogenity. J. Am. Chem. Soc. 122, 11048-11056.
  • 36. Minasov, G., Tereshko, V. & Egli, M. (1999) Atomic-resolution structures of B-DNA reveal specific influences of divalent metal ions on conformation and packing. J. Mol. Biol. 291, 83-99.
  • 37. Chiu, T.K. & Dickerson, R.E. (2000) 1 Å crystal structures of B-DNA reveal sequence-specific binding and groove-specific bending of DNA by magnesium and calcium. J. Mol. Biol. 301, 915-945.
  • 38. Halle, B. & Denisov, V.P. (1998) Water and monovalent ions in the minor groove of B-DNA oligonucleotides as seen by NMR. Biopolymers 48, 210-233.
  • 39. Denisov, V.P. & Halle, B. (2000) Sequence- specific binding of counterions to B-DNA. Proc. Natl. Acad. Sci. U.S.A. 97, 629-633.
  • 40. Hammelberg, D., McFail-Isom, L., Williams, L.D. & Wilson, W.D. (2000) Flexible structure of DNA: Ion dependence of minor-groove structure and dynamics. J. Am. Chem. Soc. 122, 10513-10520.
  • 41. Stellwagen, N.C., Magnusdottir, S., Gelfi, C. & Righetti, P.G. (2001) Preferential counterion binding to A-T tract DNA oligomers. J. Mol. Biol. 305, 1025-1033.
  • 42. Rybenkov, V.V., Vologodskii, A.V. & Cozzarelli, N.R. (1997) The effect of ionic conditions on DNA helical repeat, effective diameter and free energy of supercoiling. Nucleic Acids Res. 25, 1412-1418.
  • 43. Rybenkov, V.V., Vologodskii, A.V. & Cozzarelli, N.R. (1997) The effect of ionic conditions on the conformations of supercoiled DNA. I. Sedimentation analysis. J. Mol. Biol. 267, 299-311.
  • 44. Kao, J.Y., Goljer, I., Phan, T.A. & Bolton, P.H. (1993) Characterization of the effects of a thymine glycol residue on the structure, dynamics, and stability of duplex DNA by NMR. J. Biol. Chem. 268, 17787-17793.
  • 45. Kung, H.C. & Bolton, P.H. (1997) Structure of duplex DNA containing a thymine glycol residue in solution. J. Biol. Chem. 272, 9227- 9236.
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