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Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides.

Wang W, Wu EY, Hellinga HW, Beese LS
J Biol Chem 287 28215-26 (2012)
Related entries: 4dqi, 4dqq, 4dqr, 4dqs, 4ds4, 4ds5, 4dse, 4dsf, 4e0d

Cited: 41 times
EuropePMC logo PMID: 22648417

Abstract

In addition to discriminating against base pair mismatches, DNA polymerases exhibit a high degree of selectivity for deoxyribonucleotides over ribo- or dideoxynucleotides. It has been proposed that a single active site residue (steric gate) blocks productive binding of nucleotides containing 2'-hydroxyls. Although this steric gate plays a role in sugar moiety discrimination, its interactions do not account fully for the observed behavior of mutants. Here we present 10 high resolution crystal structures and enzyme kinetic analyses of Bacillus DNA polymerase I large fragment variants complexed with deoxy-, ribo-, and dideoxynucleotides and a DNA substrate. Taken together, these data present a more nuanced and general mechanism for nucleotide discrimination in which ensembles of intermediate conformations in the active site trap non-cognate substrates. It is known that the active site O-helix transitions from an open state in the absence of nucleotide substrates to a ternary complex closed state in which the reactive groups are aligned for catalysis. Substrate misalignment in the closed state plays a fundamental part in preventing non-cognate nucleotide misincorpation. The structures presented here show that additional O-helix conformations intermediate between the open and closed state extremes create an ensemble of binding sites that trap and misalign non-cognate nucleotides. Water-mediated interactions, absent in the fully closed state, play an important role in formation of these binding sites and can be remodeled to accommodate different non-cognate substrates. This mechanism may extend also to base pair discrimination.

Reviews - 4dqp mentioned but not cited (1)

  1. Recent progress in dissecting molecular recognition by DNA polymerases with non-native substrates. Pugliese KM, Weiss GA. Curr Opin Chem Biol 41 43-49 (2017)

Articles - 4dqp mentioned but not cited (2)

  1. Structural factors that determine selectivity of a high fidelity DNA polymerase for deoxy-, dideoxy-, and ribonucleotides. Wang W, Wu EY, Hellinga HW, Beese LS. J Biol Chem 287 28215-28226 (2012)
  2. Flexibility-rigidity index for protein-nucleic acid flexibility and fluctuation analysis. Opron K, Xia K, Burton Z, Wei GW. J Comput Chem 37 1283-1295 (2016)


Reviews citing this publication (8)

  1. Protein Ensembles: How Does Nature Harness Thermodynamic Fluctuations for Life? The Diverse Functional Roles of Conformational Ensembles in the Cell. Wei G, Xi W, Nussinov R, Ma B. Chem Rev 116 6516-6551 (2016)
  2. Ribonucleotides in DNA: origins, repair and consequences. Williams JS, Kunkel TA. DNA Repair (Amst) 19 27-37 (2014)
  3. Processing ribonucleotides incorporated during eukaryotic DNA replication. Williams JS, Lujan SA, Kunkel TA. Nat Rev Mol Cell Biol 17 350-363 (2016)
  4. The Balancing Act of Ribonucleotides in DNA. Cerritelli SM, Crouch RJ. Trends Biochem Sci 41 434-445 (2016)
  5. Sources of spontaneous mutagenesis in bacteria. Schroeder JW, Yeesin P, Simmons LA, Wang JD. Crit Rev Biochem Mol Biol 53 29-48 (2018)
  6. Building better polymerases: Engineering the replication of expanded genetic alphabets. Ouaray Z, Benner SA, Georgiadis MM, Richards NGJ. J Biol Chem 295 17046-17059 (2020)
  7. Plant Organellar DNA Polymerases Evolved Multifunctionality through the Acquisition of Novel Amino Acid Insertions. Peralta-Castro A, García-Medel PL, Baruch-Torres N, Trasviña-Arenas CH, Juarez-Quintero V, Morales-Vazquez CM, Brieba LG. Genes (Basel) 11 E1370 (2020)
  8. Bst polymerase - a humble relative of Taq polymerase. Oscorbin I, Filipenko M. Comput Struct Biotechnol J 21 4519-4535 (2023)

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  1. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ. Kent T, Chandramouly G, McDevitt SM, Ozdemir AY, Pomerantz RT. Nat Struct Mol Biol 22 230-237 (2015)
  2. DNA-protein π-interactions in nature: abundance, structure, composition and strength of contacts between aromatic amino acids and DNA nucleobases or deoxyribose sugar. Wilson KA, Kellie JL, Wetmore SD. Nucleic Acids Res 42 6726-6741 (2014)
  3. Structural basis for processivity and antiviral drug toxicity in human mitochondrial DNA replicase. Szymanski MR, Kuznetsov VB, Shumate C, Meng Q, Lee YS, Patel G, Patel S, Yin YW. EMBO J 34 1959-1970 (2015)
  4. Probing the structural and molecular basis of nucleotide selectivity by human mitochondrial DNA polymerase γ. Sohl CD, Szymanski MR, Mislak AC, Shumate CK, Amiralaei S, Schinazi RF, Anderson KS, Yin YW. Proc Natl Acad Sci U S A 112 8596-8601 (2015)
  5. Mechano-chemical kinetics of DNA replication: identification of the translocation step of a replicative DNA polymerase. Morin JA, Cao FJ, Lázaro JM, Arias-Gonzalez JR, Valpuesta JM, Carrascosa JL, Salas M, Ibarra B. Nucleic Acids Res 43 3643-3652 (2015)
  6. Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism. Gouge J, Rosario S, Romain F, Beguin P, Delarue M. J Mol Biol 425 4334-4352 (2013)
  7. Electronic measurements of single-molecule processing by DNA polymerase I (Klenow fragment). Olsen TJ, Choi Y, Sims PC, Gul OT, Corso BL, Dong C, Brown WA, Collins PG, Weiss GA. J Am Chem Soc 135 7855-7860 (2013)
  8. Structure and function of an RNA-reading thermostable DNA polymerase. Blatter N, Bergen K, Nolte O, Welte W, Diederichs K, Mayer J, Wieland M, Marx A. Angew Chem Int Ed Engl 52 11935-11939 (2013)
  9. Pre-steady-state Kinetic Analysis of a Family D DNA Polymerase from Thermococcus sp. 9°N Reveals Mechanisms for Archaeal Genomic Replication and Maintenance. Schermerhorn KM, Gardner AF. J Biol Chem 290 21800-21810 (2015)
  10. Structural accommodation of ribonucleotide incorporation by the DNA repair enzyme polymerase Mu. Moon AF, Pryor JM, Ramsden DA, Kunkel TA, Bebenek K, Pedersen LC. Nucleic Acids Res 45 9138-9148 (2017)
  11. The Closing Mechanism of DNA Polymerase I at Atomic Resolution. Miller BR, Beese LS, Parish CA, Wu EY. Structure 23 1609-1620 (2015)
  12. Processive Incorporation of Deoxynucleoside Triphosphate Analogs by Single-Molecule DNA Polymerase I (Klenow Fragment) Nanocircuits. Pugliese KM, Gul OT, Choi Y, Olsen TJ, Sims PC, Collins PG, Weiss GA. J Am Chem Soc 137 9587-9594 (2015)
  13. Steric gate residues of Y-family DNA polymerases DinB and pol kappa are crucial for dNTP-induced conformational change. Nevin P, Engen JR, Beuning PJ. DNA Repair (Amst) 29 65-73 (2015)
  14. Five checkpoints maintaining the fidelity of transcription by RNA polymerases in structural and energetic details. Wang B, Opron K, Burton ZF, Cukier RI, Feig M. Nucleic Acids Res 43 1133-1146 (2015)
  15. DNA Polymerase Conformational Dynamics and the Role of Fidelity-Conferring Residues: Insights from Computational Simulations. Meli M, Sustarsic M, Craggs TD, Kapanidis AN, Colombo G. Front Mol Biosci 3 20 (2016)
  16. Triphosphate Reorientation of the Incoming Nucleotide as a Fidelity Checkpoint in Viral RNA-dependent RNA Polymerases. Yang X, Liu X, Musser DM, Moustafa IM, Arnold JJ, Cameron CE, Boehr DD. J Biol Chem 292 3810-3826 (2017)
  17. A conservative isoleucine to leucine mutation causes major rearrangements and cold sensitivity in KlenTaq1 DNA polymerase. Wu EY, Walsh AR, Materne EC, Hiltner EP, Zielinski B, Miller BR, Mawby L, Modeste E, Parish CA, Barnes WM, Kermekchiev MB. Biochemistry 54 881-889 (2015)
  18. Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway. Chim N, Jackson LN, Trinh AM, Chaput JC. Elife 7 e40444 (2018)
  19. Prechemistry nucleotide selection checkpoints in the reaction pathway of DNA polymerase I and roles of glu710 and tyr766. Bermek O, Grindley ND, Joyce CM. Biochemistry 52 6258-6274 (2013)
  20. Landscape of π-π and sugar-π contacts in DNA-protein interactions. Wilson KA, Wells RA, Abendong MN, Anderson CB, Kung RW, Wetmore SD. J Biomol Struct Dyn 34 184-200 (2016)
  21. Kinetic mechanisms governing stable ribonucleotide incorporation in individual DNA polymerase complexes. Dahl JM, Wang H, Lázaro JM, Salas M, Lieberman KR. Biochemistry 53 8061-8076 (2014)
  22. Structural basis for the D-stereoselectivity of human DNA polymerase β. Vyas R, Reed AJ, Raper AT, Zahurancik WJ, Wallenmeyer PC, Suo Z. Nucleic Acids Res 45 6228-6237 (2017)
  23. A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA. Freund N, Taylor AI, Arangundy-Franklin S, Subramanian N, Peak-Chew SY, Whitaker AM, Freudenthal BD, Abramov M, Herdewijn P, Holliger P. Nat Chem 15 91-100 (2023)
  24. Insights into the conformation of aminofluorene-deoxyguanine adduct in a DNA polymerase active site. Vaidyanathan VG, Liang F, Beard WA, Shock DD, Wilson SH, Cho BP. J Biol Chem 288 23573-23585 (2013)
  25. Synthesis of phosphoramidate-linked DNA by a modified DNA polymerase. Lelyveld VS, Zhang W, Szostak JW. Proc Natl Acad Sci U S A 117 7276-7283 (2020)
  26. Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO-POL enzyme. Ghosh S, Goldgur Y, Shuman S. Nucleic Acids Res 48 3165-3180 (2020)
  27. Enzymatic Cleavage of 3'-Esterified Nucleotides Enables a Long, Continuous DNA Synthesis. LinWu SW, Tsai TY, Tu YH, Chi HW, Tsao YP, Chen YC, Wang HM, Chang WH, Chiou CF, Lee J, Chen CY. Sci Rep 10 7515 (2020)
  28. Primer terminal ribonucleotide alters the active site dynamics of DNA polymerase η and reduces DNA synthesis fidelity. Chang C, Lee Luo C, Eleraky S, Lin A, Zhou G, Gao Y. J Biol Chem 299 102938 (2023)
  29. A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides. Parkash V, Kulkarni Y, Bylund GO, Osterman P, Kamerlin SCL, Johansson E. Nucleic Acids Res 51 11225-11238 (2023)
  30. The enzymatic properties of Arabidopsis thaliana DNA polymerase λ suggest a role in base excision repair. Morales-Ruiz T, Beltrán-Melero C, Ortega-Paredes D, Luna-Morillo JA, Martínez-Macías MI, Roldán-Arjona T, Ariza RR, Córdoba-Cañero D. Plant Mol Biol 114 3 (2024)


Related citations provided by authors (1)

  1. Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis.. Wang W, Hellinga HW, Beese LS Proc. Natl. Acad. Sci. U.S.A. 108 17644-17648 (2011)

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