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Rat polymerase β gapped DNA interactions

Antagonistic effects of the 5′ terminal PO −4 group and magnesium on the enzyme binding to the gapped DNAs with different ssDNA gaps

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Abstract

The role of the 5′ terminal phosphate group downstream from the primer and magnesium cations in the energetics and dynamics of the gapped DNA recognition by rat polymerase β have been examined, using the fluorescence titration and stopped-flow techniques. The analyses have been performed with the entire series of gapped DNA substrates differing in the size of the ssDNA gap. The 5′ terminal phosphate group and magnesium cations exert antagonistic effect on enzyme binding to gapped DNA that depends on the length of the ssDNA gap. The PO −4 group amplifies the differences between the substrates with different ssDNA gaps, while in the presence of magnesium, affinities and structural changes induced in the DNA are very similar among examined DNA substrates. Both, the phosphate group and Mg+2 differ dramatically in affecting the thermodynamic response of the gapped DNA-rat pol β system to the salt concentration. The data indicate that these distinct effects result from affecting the structure of the DNA, in the case of the phosphate group, and from direct magnesium binding to the protein. The mechanism of rat enzyme binding depends on the length of the ssDNA gap and the presence of the 5′ terminal phosphate group. Complex formation with DNAs having three, four, and five residues in the gap occurs by a minimum three-step sequential mechanism. Depending on the presence of the 5′ terminal phosphate group and/or magnesium, binding of the enzyme to a DNA containing two residues in the ssDNA gap is described by the same three-step or by a simpler two-step mechanism. With the DNA containing only one residue in the gap, binding is always described by only a two-step mechanism. The PO −4 group and magnesium cations have opposite effects on internal stability of the complexes with different length of the ssDNA gap. While the PO −4 group increases the stability of internal intermediates with the increasing length of the gap, Mg+2 decreases the stability of the intermediates with longer ssDNA gap. As a result, the combined favorable orientation effect of the phosphate group and the unfavorable Mg+2 effect lead to the optimal docking of the ssDNA gaps with three and four residues by the enzyme.

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References

  1. Friedberg, E.C., Walker, G.C. and Siede, W. DNA Repair and Mutagenesis. ASM Press, Washington DC, 1995.

    Google Scholar 

  2. Budd, M. E. and Campbell, J. L. (1997) The roles of the eucaryotic DNA polymerases in DNA repair synthesis. Mutation Res. 384, 157–167.

    Article  PubMed  CAS  Google Scholar 

  3. Fry, M. and Loeb, L. A., Animal Cell DNA Polymerases. CRC Press, Boca Raton, FL, 1986.

    Google Scholar 

  4. Johnson, R. E., Prakash, S. and Prakash, L. (1999) Efficient bypass of a thymine-thyminedimer by yeast DNA polymerase, Pol η. Science 283, 1001–1004.

    Article  PubMed  CAS  Google Scholar 

  5. Hubscher, U., Nasheuer, H.-P. and Syvaoja, J. E. (2000) Eukaryotic DNA polymerases, a growing family. Trends Biochem. Sci. 25, 143–147.

    Article  PubMed  CAS  Google Scholar 

  6. Sobol, R.W., Horton, J.K., Kühn, R., Hua, G., Singhal, R.K., Prasad, R., et al. (1996) Requirement of mammalian DNA polymerase β in base-excision repair. Nature 379, 183–186.

    Article  PubMed  CAS  Google Scholar 

  7. Wiebauer, K. and Jiricny, J. (1990) Mismatchspecific thymine DNA glycosylase and DNA polymerase β mediate the correction of G-T mispairs in nuclear extracts from human cells. Proc. Natl. Acad. Sci. USA 87, 5842–5845.

    Article  PubMed  CAS  Google Scholar 

  8. Matsumoto, Y. and Bogenhagen, D. F. (1989) Repair of synthetic abasic site inDNA in aXenopus laevis oocyte extract. Mol. Cell. Biol. 9, 3750–3757.

    PubMed  CAS  Google Scholar 

  9. Matsumoto, Y. and Bogenhagen, D. F. (1991) Repair of a synthetic abasic site involves concerted reactions of DNA synthesis followed by excision and ligation. Mol. Cell Biol. 11, 4441–4447.

    PubMed  CAS  Google Scholar 

  10. Matsumoto, Y., Kim, K. and Bogenhagen, D. F. (1994) Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision dna repair. Mol. Cell. Biol. 14, 6187–6197.

    PubMed  CAS  Google Scholar 

  11. Prasad, R., Beard, W. A. and Wilson, S. H. (1994) Studies of gapped DNA substrate binding by mammalian dna polymerase β. J. Biol. Chem. 29 18096–18101.

    Google Scholar 

  12. Hammond, R. A., McClung, J. K. and Miller, M. R. (1990) Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N′-nitro-N-nitrosoguanidine. Biochemistry 29, 286–291.

    Article  PubMed  CAS  Google Scholar 

  13. Hoffman, J. S., Pillaire, M. J., Maga, G., Podust, V., Hubscher, U. and Villani, G. (1995) DNA polymerase β bypasses in vitro a single d(GpG)-cisplatin adduct placed on codon 13 of the HRAS gene. Proc. Natl. Acad. Sci. USA 92, 5356–5360.

    Article  Google Scholar 

  14. Masumoto, Y., and Kim, K. (1995) Excision of deoxyribose phosphate residues by DNA polymerase β during DNA repair. Science 269, 699–702.

    Article  Google Scholar 

  15. Pelletier, H., Sawaya, M. R., Kumar, A., Wilson, S. H. and Kraut, J. (1994) Structures ofternary complexes of rat DNA polymerase β, a DNA template-primer, and ddCTP. Science 264, 1891–1903.

    Article  PubMed  CAS  Google Scholar 

  16. Pelletier, H., Sawaya, M. R., Wolfle, W., Wilson, S. H. and Kraut, J. (1996) A structural basis for metal ion mutagenicity and nucleotide selectivity in human DNA polymerase β. Biochemistry 35, 12762–12777.

    Article  PubMed  CAS  Google Scholar 

  17. Sawaya, M. R., Pelletier, H., Kumar, A., Wilson, S. H. and Kraut, J. (1994) Crystal structure of rat DNA polymerase β: Evidence of a common polymerase mechanism. Science 264, 1930–1935.

    Article  PubMed  CAS  Google Scholar 

  18. Joyce, C. M. and Steitz, T. A. (1994) Function and structure relationships in DNA polymerases. Annu. Rev. Biochem. 63, 777–822.

    Article  PubMed  CAS  Google Scholar 

  19. Rajendran, S., Jezewska, M. J. and Bujalowski, W. (1998) Human DNA polymerase β recognizes single-stranded DNA using two different binding modes. J. Biol. Chem. 273, 31021–31031.

    Article  PubMed  CAS  Google Scholar 

  20. Jezewska, M. J., Rajendran, S. and Bujalowski, W. (1998) Transition between different binding modes in rat DNA polymerase β-ssDNA complexes. J. Mol. Biol. 284, 1113–1131.

    Article  PubMed  CAS  Google Scholar 

  21. Jezewska, M. J., Rajendran, S. and Bujalowski, W. (2001) Interactions of the 8-kDa domain of rat DNA polymerase β with DNA. Biochemistry 40, 3295–3307.

    Article  PubMed  CAS  Google Scholar 

  22. Jezewska, M. J., Rajendran, S. and Bujalowski, W. (2001) Energetics and specificity of rat DNA polymerase β interactions with template-primer and gapped DNA substrates. J. Biol. Chem. 276, 16123–16136.

    Article  PubMed  CAS  Google Scholar 

  23. Rajendran, S., Jezewska, M.J. and Bujalowski, W. (2001) Recognition of template primer and gapped DNA substrates by human DNA polymerase β. J. Mol. Biol. 308, 477–500.

    Article  PubMed  CAS  Google Scholar 

  24. Jezewska, M. J., Galletto, R. and Bujalowski, W. (2002) Dynamics of gapped DNA recognition by human polymerase β. J. Biol. Chem. 277, 20316–20327.

    Article  PubMed  CAS  Google Scholar 

  25. Jezewska, M. J., Rajendran, S., Galletto, R. and Bujalowski, W. (2001) Kinetic mechanisms of rat polymerase β-ssDNA interactions. Quantitative fluorescence stopped-flow analysis of the formation of the (Pol β)16 and (Pol β)5 binding mode. J. Mol. Biol. 313, 977–1002.

    Article  PubMed  CAS  Google Scholar 

  26. Edelhoch, H. (1967) Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954.

    Article  PubMed  CAS  Google Scholar 

  27. Gill, S. C. and von Hippel, P. H. (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal. Biochem. 182, 319–326.

    Article  PubMed  CAS  Google Scholar 

  28. Bujalowski, W., Klonowska, M. M. and Jezewska, M. J. (1994) Oligomeric structure of Escherichia coli primary replicative helicase DnaB protein. J. Biol. Chem. 269, 1–9.

    Google Scholar 

  29. Galletto, R., Rajendran, S., and Bujalowski, W. (2000) Interactions of nucleotide cofactors with the Escherichia coli replication factor DnaC protein. Biochemistry 39, 12959–12969.

    Article  PubMed  CAS  Google Scholar 

  30. Bujalowski, W. and Jezewska, M. J. (1995) Interactions of Escherichia coli primary replicative helicase DnaB protein with single-stranded DNA. The nucleic acid does not wrap around the protein hexamer. Biochemistry 34, 8513–8519.

    Article  PubMed  CAS  Google Scholar 

  31. Jezewska, M. J. and Bujalowski, W. (1996) Global conformational transitions in E. coli primary replicative DnaB protein induced by ATP, ADP and single-stranded DNA binding. J. Biol. Chem. 271, 4261–4265.

    Article  PubMed  CAS  Google Scholar 

  32. Jezewska, M. J., Rajendran, S., Bujalowska, D. and Bujalowski, W. (1998) Does ssDNA pass through the inner channel of the protein hexamer in the complex with the E. coli dnaB helicase? Fluorescence energy transfer studies. J. Biol. Chem. 273, 10515–10529.

    Article  PubMed  CAS  Google Scholar 

  33. Jezewska, M. J., Rajendran, S. and Bujalowski, W. (1998) Functional and structural heterogeneity of the DNA binding of the E. coli primary replicative helicase DnaB protein. J. Biol. Chem. 273, 9058–9069.

    Article  PubMed  CAS  Google Scholar 

  34. Jezewska, M. J., Rajendran, S. and Bujalowski, W. (1997) Complex of Escherichia coli primary replicative helicase DnaB protein with a replication fork. Recognition and structure. Biochemistry 37, 3116–3136.

    Article  Google Scholar 

  35. Bujalowski, W. and Jezewska, M. J. Spectrophotometry & Spectrofluorimetry. A Practical Approach. (Gore, M. G., ed.) Oxford University Press, London, 2000.

    Google Scholar 

  36. Bujalowski, W. and Jezewska, M. J. (2000) Kinetic mechanism of the single-stranded DNA recognition by Escherichia coli replicative helicase DnaB protein. Application of the matrix projection operator technique to analyze stopped-flow kinetics. J. Mol. Biol. 295, 831–852.

    Article  PubMed  CAS  Google Scholar 

  37. Bujalowski, W. and Jezewska, M. J. (2000) Kinetic mechanism of nucleotide cofactor binding to Escherichia coli replicative helicase DnaB protein. Stopped-flow kinetic studies using fluorescent ribose-, and base-modified nucleotide analogs. Biochemistry 39, 2106–2122.

    Article  PubMed  CAS  Google Scholar 

  38. Rajendran, S., Jezewska, M. J., and Bujalowski, W. (2000) Multiple-step kinetic mechanism of DNA-independent ATP binding and hydrolysis by Escherichia coli replicative helicase DnaB protein: Quantitative analysis using the rapid quench-flow method. J. Mol. Biol. 303, 773–795.

    Article  PubMed  CAS  Google Scholar 

  39. Pilar, R. L. (1968) Elementary Quantum Chemistry. New York: McGraw-Hill.

    Google Scholar 

  40. Record, M. T., Lohman, T. M., and deHaseth, P. L. (1976) Ion effects on ligand-nucleic acid interactions. J. Mol. Biol. 107, 145–158.

    Article  PubMed  CAS  Google Scholar 

  41. Record, M. T., Jr., Anderson, C. F., and Lohman, T. M. (1978) Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: the roles of ion association or release, screening, and ion effects on water activity. Q. Rev. Biophys. 11, 103–178.

    Article  PubMed  CAS  Google Scholar 

  42. Von Hippel, P. H. and Schleich, T. Structure of Biological Macromolecules. Timasheff, S. and Fasman G. D. Eds. New York: M. Dekker, New York, 1969.

    Google Scholar 

  43. Bernasconi, C.J. Relaxation Kinetics. Academic Press, NY, 1976.

    Google Scholar 

  44. Bujalowski, W., Greaser, E., McLaughlin, L.W. and Porschke, D. (1986) Anticodon loop of tRNAPhe: structure, dynamics and Mg+2 binding. Biochemistry 25, 6365–6371.

    Article  PubMed  CAS  Google Scholar 

  45. Bujalowski, W., Jung, M., McLaughlin, L. W. and Porschke, D. (1986) Codon-induced association of the isolated anticodon loop of tRNAPhe. Biochemistry 25, 6372–6378.

    Article  PubMed  CAS  Google Scholar 

  46. Jezewska, M. J., Galletto, R. and Bujalowski, W. (2001) Multiple-step kinetic mechanisms of the ssDNA recognition process by human polymerase β in its different binding modes. Biochemistry 40, 11794–11810.

    Article  PubMed  CAS  Google Scholar 

  47. Connors, K.W. Chemical Kinetics. The Study of Reaction Rates in Solution VCH, New York, 1990, pp. 133–186.

    Google Scholar 

  48. Berry, R.S., Rice, S.A. and Ross, J. Physical Chemistry. New York: Wiley, New York, 1980, pp. 1117–1204.

    Google Scholar 

  49. Porschke, D. (1976) The nature of stacking interactions in polynucleotides. Molecular states in oligo-polribocytidylic acids by relaxation analysis. Biochemistry 15, 1495–1499.

    Article  Google Scholar 

  50. Porschke, D. (1978) Molecular states in singlestranded adenylate chains by relaxation analysis, Biopolymers 17, 315–232.

    Article  Google Scholar 

  51. Zhong, X., Patel, S. S., Werneburg, B. G. and Tsai, M-D. (1997) DNA polymerase β: conformational changes in the mechanism of catalysis. Biochemistry 36, 11891–11900.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Department of Human Biological Chemistry and Genetics and The Sealy Center for Structural Biology, The University of Texas Medical Branch at Galveston, 77555-1053, Galveston, TX

    Maria J. Jezewska, Roberto Galletto & Wlodzimiez Bujalowski

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  1. Maria J. Jezewska
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  2. Roberto Galletto
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  3. Wlodzimiez Bujalowski
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Correspondence to Wlodzimiez Bujalowski.

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This work was supported by NIH Grant GM-58565 (to W. B.)

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Jezewska, M.J., Galletto, R. & Bujalowski, W. Rat polymerase β gapped DNA interactions. Cell Biochem Biophys 38, 125–160 (2003). https://doi.org/10.1385/CBB:38:2:125

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