Theoretical Chemistry Accounts

, 131:1287 | Cite as

Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms

  • Tamar Schlick
  • Karunesh Arora
  • William A. Beard
  • Samuel H. Wilson
Regular Article

Abstract

In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.

Keywords

Enzyme catalysis Intrinsic protein dynamics Pre-chemistry conformational adjustments Nucleotidyl transfer DNA polymerase β Catalytic cycle chemical step 

Notes

Acknowledgments

Research described in this Article was supported in part by Philip Morris USA Inc. and Philip Morris International and by NSF award MCB-0316771, and NIH award R01 ES012692 to T.S., and Research Project Numbers Z01-ES050158 and Z01-ES050161 to S.H.W. in the Intramural Research Program of the National Institutes of Health, National Institute of Environmental Health Sciences and was in association with NIH award 1U19CA105010.

References

  1. 1.
    Garcia-Viloca M, Gao J, Karplus M, Truhlar DG (2004) How enzymes work: analysis by modern rate theory and computer simulations. Science 303:186CrossRefGoogle Scholar
  2. 2.
    Kamerlin SCL, Warshel A (2010) At the dawn of the 21st century: is dynamics the missing link for understanding enzyme catalysis? Proteins 78:1339Google Scholar
  3. 3.
    Karplus M (2010) Role of conformation transitions in adenylate kinase. Proc Natl Acad Sci USA 107:E71CrossRefGoogle Scholar
  4. 4.
    McGeagh JD, Ranaghan KE, Mulholland AJ (2011) Protein dynamics and enzyme catalysis: insights from simulations. Biochim Biophys Acta 1814:1077CrossRefGoogle Scholar
  5. 5.
    Nagel ZD, Klinman JP (2006) Tunneling and dynamics in enzymatic hydride transfer. Chem Rev 106:3095CrossRefGoogle Scholar
  6. 6.
    Nashine VC, Hammes-Schiffer S, Benkovic SJ (2010) Coupled motions in enzyme catalysis. Curr Opin Chem Biol 14:644CrossRefGoogle Scholar
  7. 7.
    Villali J, Kern D (2010) Choreographing an enzyme’s dance. Curr Opin Chem Biol 14:636CrossRefGoogle Scholar
  8. 8.
    Hammes GG, Benkovic SJ, Hammes-Schiffer S (2011) Flexibility, diversity, and cooperativity: pillars of enzyme catalysis. Biochemistry 50:10422CrossRefGoogle Scholar
  9. 9.
    Beard WA, Wilson SH (2006) Structure and mechanism of DNA polymerase β. Chem Rev 106:361CrossRefGoogle Scholar
  10. 10.
    Beard WA, Wilson SH (2003) Structural insights into the origins of DNA polymerase fidelity. Structure (Camb) 11:489CrossRefGoogle Scholar
  11. 11.
    Joyce CM, Benkovic SJ (2004) DNA polymerase fidelity: kinetics, structure, and checkpoints. Biochemistry 43:14317CrossRefGoogle Scholar
  12. 12.
    Batra VK, Beard WA, Shock DD, Krahn JM, Pedersen LC, Wilson SH (2006) Magnesium induced assembly of a complete DNA polymerase catalytic complex. Structure (Camb) 14:757CrossRefGoogle Scholar
  13. 13.
    Kirby TW, DeRose EF, Cavanaugh NA, Beard WA, Shock DD, Mueller GA, Wilson SH, London RE (2012) Metal-induced DNA translocation leads to DNA polymerase conformational activation. Nucl Acids Res 40:2974CrossRefGoogle Scholar
  14. 14.
    Prasad BR, Warshel A (2011) Prechemistry versus preorganization in DNA replication fidelity. Proteins 79:2900CrossRefGoogle Scholar
  15. 15.
    Sawaya MR, Prasad P, Wilson SH, Kraut J, Pelletier H (1997) Crystal structures of human DNA polymerase β complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry 36:11205CrossRefGoogle Scholar
  16. 16.
    Zhong X, Patel SS, Tsai M-D (1998) DNA polymerase β. 5. Dissecting the functional roles of the two metal ions with Cr(III)dTTP. J Am Chem Soc 120:235CrossRefGoogle Scholar
  17. 17.
    Radhakrishnan R, Schlick T (2005) Fidelity discrimination in DNA polymerase β: differing closing profiles for a mismatched (G:A) versus matched (G:C) base pair. J Am Chem Soc 127:13245CrossRefGoogle Scholar
  18. 18.
    Lin P, Pedersen LC, Batra VK, Beard WA, Wilson SH, Pedersen LG (2006) Energy analysis of chemistry for correct insertion by DNA polymerase β. Proc Natl Acad Sci USA 103:13294CrossRefGoogle Scholar
  19. 19.
    Lin P, Batra VK, Pedersen LC, Beard WA, Wilson SH, Pedersen LG (2008) Incorrect nucleotide insertion at the active site of a G:A mismatch catalyzed by DNA polymerase β. Proc Natl Acad Sci USA 105:5670CrossRefGoogle Scholar
  20. 20.
    Radhakrishnan R, Schlick T (2006) Correct and incorrect nucleotide incorporation pathways in DNA polymerase β. Biochem Biophys Res Commun 350:521CrossRefGoogle Scholar
  21. 21.
    Wang Y, Schlick T (2008) Quantum mechanics/molecular mechanics investigation of the chemical reaction in Dpo4 reveals water-dependent pathways and requirements for active site reorganization. J Am Chem Soc 130:13240CrossRefGoogle Scholar
  22. 22.
    Koshland DE Jr (1958) Application of a theory of enzyme specificity to protein synthesis. Proc Natl Acad Sci USA 44:98CrossRefGoogle Scholar
  23. 23.
    Radhakrishnan R, Arora K, Wang Y, Beard WA, Wilson SH, Schlick T (2006) Regulation of DNA repair fidelity by molecular checkpoints: “Gates” in DNA polymerase β’s substrate selection. Biochemistry 45:15142CrossRefGoogle Scholar
  24. 24.
    Radhakrishnan R, Schlick T (2004) Orchestration of cooperative events in DNA synthesis and repair mechanism unraveled by transition path sampling of DNA polymerase β’s closing. Proc Natl Acad Sci USA 101:5970CrossRefGoogle Scholar
  25. 25.
    Yang L, Arora K, Beard WA, Wilson SH, Schlick T (2004) Critical role of magnesium ions in DNA polymerase β’s closing and active site assembly. J Am Chem Soc 126:8441CrossRefGoogle Scholar
  26. 26.
    Yang L, Beard W, Wilson S, Roux B, Broyde S, Schlick T (2002) Local deformations revealed by dynamics simulations of DNA polymerase β with DNA mismatches at the primer terminus. J Mol Biol 321:459CrossRefGoogle Scholar
  27. 27.
    Yang L, Beard WA, Wilson SH, Broyde S, Schlick T (2002) Polymerase β simulations suggest that Arg258 rotation is a slow step rather than large subdomain motions per se. J Mol Biol 317:679CrossRefGoogle Scholar
  28. 28.
    Yang L, Beard WA, Wilson SH, Broyde S, Schlick T (2004) Highly organized but pliant active site of DNA polymerase β: compensatory mechanisms in mutant enzymes revealed by dynamics simulations and energy analyses. Biophysical J 86:3392CrossRefGoogle Scholar
  29. 29.
    Arora K, Brooks CL III (2007) Large-scale allosteric conformational transitions of adenylate kinase appear to involve a population-shift mechanism. Proc Natl Acad Sci USA 104:18496CrossRefGoogle Scholar
  30. 30.
    Bucher D, Grant BJ, McCammon JA (2011) Induced fit or conformational selection? The role of the semi-closed state in the maltose binding protein. Biochemistry 50:10530CrossRefGoogle Scholar
  31. 31.
    Hammes GG, Chang Y-C, Oas TG (2009) Conformational selection or induced fit: a flux description of reaction mechanism. Proc Natl Acad Sci USA 106:13737CrossRefGoogle Scholar
  32. 32.
    Ma B, Nussinov R (2010) Enzyme dynamics point to stepwise conformational selection in catalysis. Curr Opin Chem Biol 14:652CrossRefGoogle Scholar
  33. 33.
    Wlodarski T, Zagrovic B (2009) Conformational selection and induced fit mechanism underlie specificity in noncovalent interactions with ubiquitin. Proc Natl Acad Sci USA 106:19346CrossRefGoogle Scholar
  34. 34.
    Foley MC, Arora K, Schlick T (2012) Intrinsic motions of DNA polymerases underlie their remarkable specificity and selectivity and suggest a hybrid substrate binding mechanism. In: Schlick T (ed) Innovations in bimolecular modeling and simulations, vol 2., Royal Society of ChemistryLondon, UK, pp 81–110CrossRefGoogle Scholar
  35. 35.
    Arora K, Brooks III CL, Schlick T (2012) Computational support for hybrid induced fit and conformational selection mechanisms. In preparationGoogle Scholar
  36. 36.
    Santoso Y, Joyce CM, Potapova O, Le Reste L, Hohlbein J, Torella JP, Grindley NDF, Kapanidis AN (2010) Conformational transitions in DNA polymerase I revealed by single-molecule FRET. Proc Natl Acad Sci USA 107:715CrossRefGoogle Scholar
  37. 37.
    Rothwell PJ, Berger S, Kensch O, Felekyan S, Antonik M, Wöhrl BM, Restle T, Goody RS, Seidel CAM (2003) Multiparameter single-molecule fluorescence spectroscopy reveals heterogeneity of HIV-1 reverse transcriptase:primer/template complexes. Proc Natl Acad Sci USA 100:1655CrossRefGoogle Scholar
  38. 38.
    Arora K, Brooks CL III (2009) Functionally important conformations of the Met20 loop in dihydrofolate reductase are populated by rapid thermal fluctuations. J Am Chem Soc 131:5642CrossRefGoogle Scholar
  39. 39.
    Bhabha G, Lee J, Ekiert DC, Gam J, Wilson IA, Dyson HJ, Benkovic SJ, Wright PE (2011) A dynamic knockout reveals that conformational fluctuations influence the chemical step of enzyme catalysis. Science 332:234CrossRefGoogle Scholar
  40. 40.
    Eisenmesser EZ, Millet O, Labeikovsky W, Korzhnev DM, Wolf-Watz M, Bosco DA, Skalicky JJ, Kay LE, Kern D (2005) Intrinsic dynamics of an enzyme underlies catalysis. Nature 438:117CrossRefGoogle Scholar
  41. 41.
    Johnson KA (2008) Role of induced fit in enzyme specificity: a molecular forward/reverse switch. J Biol Chem 283:26297CrossRefGoogle Scholar
  42. 42.
    Beard WA, Shock DD, Vande Berg BJ, Wilson SH (2002) Efficiency of correct nucleotide insertion governs DNA polymerase fidelity. J Biol Chem 277:47393CrossRefGoogle Scholar
  43. 43.
    Pelletier H, Sawaya MR, Kumar A, Wilson SH, Kraut J (1994) Structures of ternary complexes of rat DNA polymerase β, a DNA template-primer, and ddCTP. Science 264:1891CrossRefGoogle Scholar
  44. 44.
    Beard WA, Osheroff WP, Prasad R, Sawaya MR, Jaju M, Wood TG, Kraut J, Kunkel TA, Wilson SH (1996) Enzyme-DNA interactions required for efficient nucleotide incorporation and discrimination in human DNA polymerase β. J Biol Chem 271:12141CrossRefGoogle Scholar
  45. 45.
    Iwanaga A, Ouchida M, Miyazaki K, Hori K, Mukai T (1999) Functional mutation of DNA polymerase β found in human gastric cancer—inability of the base excision repair in vitro. Mutat Res 435:121CrossRefGoogle Scholar
  46. 46.
    Lang T, Dalal S, Chikova A, DiMaio D, Sweasy JB (2007) The E295K DNA polymerase beta gastric cancer-associated variant interferes with base excision repair and induces cellular transformation. Mol Cell Biol 27:5587CrossRefGoogle Scholar
  47. 47.
    Li Y, Gridley CL, Jaeger J, Sweasy JB, Schlick T (2012) Unfavorable electrostatic and steric interactions in DNA polymerase β E295K mutant interfere with the enzyme’s pathway. J Am Chem Soc 134:9999CrossRefGoogle Scholar
  48. 48.
    Batra VK, Beard WA, Shock DD, Pedersen LC, Wilson SH (2008) Structures of DNA polymerase β with active site mismatches suggest a transient abasic site intermediate during misincorporation. Mol Cell 30:315CrossRefGoogle Scholar
  49. 49.
    Bryant FR, Johnson KA, Benkovic SJ (1983) Elementary steps in the DNA polymerase I pathway. Biochemistry 22:3537CrossRefGoogle Scholar
  50. 50.
    Joyce CM, Potapova O, DeLucia AM, Huang X, Basu VP, Grindley NDF (2008) Fingers-closing and other rapid conformational changes in DNA polymerase I (Klenow fragment) and their role in nucleotide selectivity. Biochemistry 47:6103CrossRefGoogle Scholar
  51. 51.
    Rothwell PJ, Mitaksov V, Waksman G (2005) Motions of the fingers subdomain of Klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases. Mol Cell 19:345CrossRefGoogle Scholar
  52. 52.
    Tsai Y-C, Johnson KA (2006) A new paradigm for DNA polymerase specificity. Biochemistry 45:9675CrossRefGoogle Scholar
  53. 53.
    Kirmizialtin S, Nguyen V, Johnson KA, Elber R (2012) How conformational dynamics of DNA polymerase select correct substrates: experiments and simulations. Structure (Camb) 20:618CrossRefGoogle Scholar
  54. 54.
    Zhang H, Cao W, Zakharova E, Konigsberg W, De La Cruz EM (2007) Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates. Nucl Acids Res 35:6052CrossRefGoogle Scholar
  55. 55.
    Hanes JW, Johnson KA (2007) A novel mechanism of selectivity against AZT by the human mitochondrial DNA polymerase. Nucl Acids Res 35:6973CrossRefGoogle Scholar
  56. 56.
    Post CB, Ray WJ Jr (1995) Reexamination of induced fit as a determinant of substrate specificity in enzymatic reactions. Biochemistry 34:15881CrossRefGoogle Scholar
  57. 57.
    Mulholland A, Roitberg AE, Tuñón I (2012) Enzyme dynamics and catalysis in the mechanism of DNA polymerase. Theor Chem Acc 131:1286CrossRefGoogle Scholar
  58. 58.
    Schlick T, Collepardo-Guevara R, Halvorsen LA, Jung S, Xiao X (2011) Biomolecular modeling and simulation: a field coming of age. Q Rev Biophys 44:191CrossRefGoogle Scholar
  59. 59.
    Wang Y, Reddy S, Beard WA, Wilson SH, Schlick T (2007) Differing conformational pathways before and after chemistry for insertion of dATP versus dCTP opposite 8-oxoG in DNA polymerase β. Biophys J 92:3063CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Tamar Schlick
    • 1
    • 2
  • Karunesh Arora
    • 3
  • William A. Beard
    • 4
  • Samuel H. Wilson
    • 4
  1. 1.Department of ChemistryNew York UniversityNew YorkUSA
  2. 2.Courant Institute of Mathematical SciencesNew York UniversityNew YorkUSA
  3. 3.Departments of Chemistry and BiophysicsUniversity of MichiganAnn ArborUSA
  4. 4.Laboratory of Structural BiologyNational Institute of Environmental Sciences, National Institutes of HealthResearch Triangle ParkUSA

Personalised recommendations