Nucleotide Analogues as Probes for DNA and RNA Polymerases

Robert D. Kuchta1

1 University of Colorado, Boulder, Colorado
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch090203
Online Posting Date:  June, 2010
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Nucleotide analogues represent a major class of anti‐cancer and anti‐viral drugs, and provide an extremely powerful tool for dissecting the mechanisms of DNA and RNA polymerases. While the basic assays themselves are relatively straightforward, a key issue is to appropriately design the studies to answer the mechanistic question of interest. This unit addresses the major issues involved in designing these studies, and some of the potential difficulties that arise in interpreting the data. Examples are given for the type of analogues typically used, the experimental approaches with different polymerases, and issues with data interpretation. Curr. Protoc. Chem Biol. 2:111‐124. © 2010 by John Wiley & Sons, Inc.

Keywords: polymerase; nucleotide; DNA; RNA; kinetics

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Table of Contents

  • Introduction
  • Sugar Analogues
  • Base Analogues
  • Effects of DNA Sequence
  • 3′‐5′ Exonuclease Activity
  • Challenges of RNA Polymerases
  • Data Interpretation
  • Examples of Difficulty Interpreting how a Polymerase Interacts with a Nucleotide
  • Acknowledgements
  • Literature Cited
  • Figures
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Literature Cited

Literature Cited
   Anand, V.S. and Patel, S.S. 2006. Transient state kinetics of transcription elongation by T7 RNA polymerase. J. Biol. Chem. 281:35677‐35685.
   Beckman, J., Kincaid, K., Hocek, M., Spratt, T., Engels, J., Cosstick, R., and Kuchta, R.D. 2007. Human DNA polymerase alpha uses a combination of positive and negative selectivity to polymerize purine dNTPs with high fidelity. Biochemistry 46:448‐460.
   Bell, J.B., Eckert, K.A., Joyce, C.M., and Kunkel, T.A. 1997. Base miscoding and strand misalignment errors by mutator Klenow polymerases with amino acid substitutions at tyrosine 766 in the O‐helix of the Fingers subdomain. J. Biol. Chem. 272:7345‐7351.
   Berdis, A.J. and McCutcheon, D. 2007. The use of non‐natural nucleotides to probe template‐independent DNA synthesis. Chembiochem 8:1399‐1408.
   Boosalis, M.S., Petruska, J., and Goodman, M.F. 1987. DNA polymerase insertion fidelity: Gel assay for site‐specific kinetics. J. Biol. Chem. 262:14689‐14696.
   Burgers, P.M., Koonin, E.V., Bruford, E., Blanco, L., Burtis, K.C., Christman, M.F., Copeland, W.C., Friedberg, E.C., Hanaoka, F., Hinkle, D.C., Lawrence, C.W., Nakanishi, M., Ohmori, H., Prakash, L., Prakash, S., Reynaud, C.A., Sugino, A., Todo, T., Wang, Z., Weill, J.C., and Woodgate, R. 2001. Eukaryotic DNA polymerases: Proposal for a revised nomenclature. J. Biol. Chem. 276:43487‐43490.
   Burgess, K. and Cook, D. 2000. Syntheses of nucleoside triphosphates. Chem. Rev. 100: 2047‐2060.
   Burton, J.R. and Everson, G.T. 2009. HCV NS5B polymerase inhibitors. Curr. Liver Dis. 13:453‐465.
   Cai, H., Bloom, L.B., Eritja, R., and Goodman, M.F. 1993. Kinetics of deoxyribonucleotide insertion and extension at abasic template lesions in different sequence contexts using HIV‐1 reverse transcriptase. J. Biol. Chem. 268:23567‐23572.
   Castro, C., Arnold, J.J., and Cameron, C.E. 2005. Incorporation fidelity of the viral RNA‐dependent RNA polymerase: A kinetic, thermodynamic and structural perspective. Virus Res. 107:141‐149.
   Cavanaugh, N.A., Urban, M., Beckman, J., Spratt, T., and Kuchta, R.D. 2009. Identifying the features of purine dNTPs that allow accurate and efficient DNA synthesis by herpes simplex virus 1 DNA polymerase. Biochemistry 48:3554‐3564.
   Chiaramonte, M., Moore, C.L., Kincaid, K., and Kuchta, R.D. 2003. Facile polymerization of dNTPs bearing unnatural base analogues by DNA polymerase alpha and Klenow fragment (DNA polymerase I). Biochemistry 42:10472‐10481.
   Choi, D.J., Roth, R.B., Liu, T., Geacintov, N.E., and Scicchitano, D.A. 1996. Incorrect base insertion and prematurely terminated transcripts during T7 RNA polymerase Transcription elongation past benzo[a]pyrenediol epoxide‐modified DNA. J. Mol. Biol. 264:123‐219.
   Dahlberg, M.E. and Benkovic, S.J. 1991. Kinetic mechanism of DNA polymerase I (Klenow fragment): Identification of a second conformational change and evaluation of the internal equilibrium constant. Biochemistry 30:4835‐4843.
   Donlin, M.J., Patell, S.S., and Johnson, K.A. 1991. Kinetic partitioning between the exonuclease and polymerase sites in DNA error correction. Biochemistry 30:538‐547.
   Hatoum, A. and Roberts, J. 2008. Prevalence of RNA polymerase stalling at Escherichia coli promoters after open complex formation. Mol. Microbiol. 68:17‐28.
   Hendrickson, C.L., Devine, K.G., and Benner, S.A. 2004. Probing minor groove recognition contacts by DNA polymerases and reverse transcriptases using 3‐deaza‐2'‐deoxyadenosine. Nucl. Acids Res. 32:2241‐2250.
   Henry, A.A., Yu, C., and Romesberg, F.E. 2003. Determinants of unnatural nucleobase stability and polymerase recognition. J. Am. Chem. Soc. 125:9638‐9646.
   Henry, A.A., Olsen, A.G., Matsuda, S., Yu, C., Geierstanger, B.H., and Romesberg, F.E. 2004. Efforts to expand the genetic alphabet: Identification of a replicable unnatural DNA self‐pair. J. Am. Chem. Soc. 126:6923‐6931.
   Hieb, A.R., Baran, S., Goodrich, J.A., and Kugel, J.F. 2006. An 8 Nt. RNA triggers a rate‐limiting shift of RNA polymerase II complexes into elongation. EMBO J. 25:3100‐3109.
   Hirao, I., Kimoto, M., Mitsui, T., Fujiwara, T., Kawai, R., Sato, A., Harada, Y., and Yokoyama, S. 2006. An unnatural hydrophobic base pair system: Site‐specific incorporation of nucleotide analogs into DNA and RNA. Nat. Methods 3:729‐735.
   Hirao, I., Mitsui, T., Kimoto, M., and Yokoyama, S. 2007. An efficient unnatural base pair for PCR amplification. J. Am. Chem. Soc. 129:15549‐15555.
   Horwitz, J.P., Chua, J., Noel, M., and DaRooge, M.A. 1964. Nucleosides. IV. 1‐(2‐deoxy‐beta‐D‐lyxofuranosyl)‐5‐iodouracil. J. Med. Chem. 7:385‐386.
   Hsieh, J.C., Zinnen, S., and Modrich, P. 1993. Kinetic mechanism of the DNA‐dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. J. Biol. Chem. 268:24607‐24613.
   Huang, P., Chubb, S., Hertel, L.W., Grindey, G.B., and Plunkett, W. 1991. Action of 2′,2′‐difluorocytidine on DNA synthesis. Canc. Res. 51:6110‐6117.
   Hwang, G.T. and Romesberg, F.E. 2008. Unnatural substrate repertoire of A, B, and X family DNA polymerases. J. Am. Chem. Soc. 130:14872‐14882.
   Kim, T.W., Delaney, J.C., Essigmann, J.M., and Kool, E.T. 2005. Probing the active site tightness of DNA polymerase in subangstrom increments. Proc. Nat. Acad. Sci. U.S.A. 102:15803‐15808.
   Kincaid, K., Beckman, J., Zivkovic, A., Halcomb, R.L., Engels, J., and Kuchta, R.D. 2005. Exploration of factors driving incorporation of unnatural dNTPs into DNA by Klenow fragment (DNA polymerase I) and DNA polymerase alpha. Nucl. Acids Res. 33:2620‐2628.
   Kool, E.T. 2002. Active site tightness and substrate fit in DNA replication. Ann. Rev. Biochem. 71:191‐219.
   Kuchta, R.D. and Willhelm, L. 1991. Inhibition of DNA primase by 9‐beta‐D‐arabinofuranosyladenosine triphosphate. Biochemistry 30:797‐803.
   Kuchta, R.D., and Stengel, G. 2009. Mechanism and evolution of DNA primases. Biochim. Biophys. Acta. In press.
   Kuchta, R.D., Mizrahi, V., Benkovic, P.A., Johnson, K.A., and Benkovic, S.J. 1987. Kinetic mechanism of DNA polymerase I (Klenow). Biochemistry 26:8410‐8417.
   Kuchta, R.D., Benkovic, P., and Benkovic, S.J. 1988. Kinetic mechanism whereby DNA polymerase I (Klenow) replicates DNA with high fidelity. Biochemistry 27:6716‐6725.
   Kuchta, R.D., Ilsley, D., Kravig, K.D., Schubert, S., and Harris, B. 1992. Inhibition of DNA primase and polymerase alpha by arabinofuranosylnucleoside triphosphates and related compounds. Biochemistry 31:4720‐4728.
   Kunkel, T.A. 1985. The mutational specificity of DNA polymerase beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J. Biol. Chem. 260:5787‐5796.
   Kunkel, T.A. and Alexander, P.S. 1986. The base substitution fidelity of eukaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J. Biol. Chem. 261:160‐166.
   Lai, M.D. and Beattie, K.L. 1988. Influence of DNA sequence on the nature of mispairing during DNA synthesis. Biochemistry 27:1722‐1728.
   Leconte, A.M., Hwang, G.T., Matsuda, S., Capek, P., Hari, Y., and Romesberg, F.E. 2008. Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet. J. Am. Chem. Soc. 130:2336‐2343.
   Lee, I. and Berdis, A.J. 2009. Non‐natural nucleotides as probes for the mechanism and fidelity of DNA polymerases. Biochim. Biophys. Acta. Epub ahead of print.
   Lee, J.R., Helquist, S.A., Kool, E.T., and Johnson, K.A. 2008. Importance of hydrogen bonding for efficiency and specificity of the human mitochondrial DNA polymerase. J. Biol. Chem. 283:14402‐14410.
   Lin, T.S. and Mancini, W.R. 1983. Synthesis and antineoplastic activity of 3′‐azido and 3′‐amino analogs of pyrimidine deoxyribonuclosides. J. Med. Chem. 26:544‐588.
   Ludwig, J. 1981. A new route to nucleoside 5′‐triphosphates. Acta Biochim. Biophys. Acad. Sci. Hung. 16:131‐135.
   Lutz, M.J., Held, H.A., Hottinger, M., Hubscher, U., and Benner, S.A. 1996. Differential discrimination of DNA polymerases for variants of the non‐standard nucleobase pair between xanthosine and 2,4‐diaminopyrimidine, two components of an expanded genetic alphabet. Nucl. Acids Res. 24:1308‐1313.
   Matsuda, S., Henry, A.A., Schultz, S.S., and Romesberg, F.E. 2003. The effects of minor‐groove hydrogen‐bond acceptors and donors on the stability and replication of four unnatural base pairs. J. Am. Chem. Soc. 125:6135‐6139.
   Meyer, A.S., Blandino, M., and Spratt, T. 2004. E. coli DNA polymerase I (Klenow fragment) uses a hydrogen‐bonding fork from Arg668 to the primer terminus and incoming dNTP to catalyze DNA replication. J. Biol. Chem. 279:33043‐33046.
   Moore, C.L., Zivkovic, A., Engels, J., and Kuchta, R.D. 2004. Human DNA primase uses Watson‐Crick hydrogen bonding groups to distinguish between correct and incorrect NTPs. Biochemistry 43:12367‐12374.
   Morales, J.C. and Kool, E.T. 1998. Efficient replication between non‐hydrogen bonded nucleoside shape analogs. Nat. Struct. Biol. 5:950‐954.
   Morales, J.C. and Kool, E.T. 2000. Varied molecular interactions at the active sites of several DNA polymerases: Nonpolar nucleoside isosteres as probes. J. Am. Chem. Soc. 122:1001‐1007.
   Moran, S., Ren, R.X.‐F., Rumney, S., and Kool, E.T. 1997. Difluorotoluene, a nonpolar isostere of thymine, codes specifically and efficiently for adenine in DNA replication. J. Am. Chem. Soc. 119:2056‐2057.
   Ogawa, A.K., Wu, Y., McMinn, D.L., Liu, J., Schultz, P.G., and Romesberg, F.E. 2000. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. J. Am. Chem. Soc. 122:3274‐3287.
   Parker, W. 2009. Enzymology of purine and pyrimidine antimetabolites in the treatment of cancer. Chem. Rev. 109:2880‐2893.
   Patel, S.S., Wong, I., and Johnson, K.A. 1991. Pre‐steady state kinetic analysis of processive DNA replication including complete characterization of an exonuclease deficient mutant. Biochemistry 30:511.
   Patro, J.N., Urban, M., and Kuchta, R.D. 2009. Role of the 2‐amino group of purines during dNTP polymerization by human DNA polymerase alpha. Biochemistry 48:180‐189.
   Piccirilli, J.A., Krauch, T., Moroney, S.E., and Benner, S.A. 1990. Enzymatic incorporation of a new base pair into DNA and RNA extends the genetic alphabet. Nature 343:33‐37.
   Plunkett, W., Huang, P., Searcy, C.E., and Gandhi, V. 1996. Gemcitabine: Preclinical pharmacology and mechanisms of action. Semin. Oncol. 56:3‐15.
   Ramirez‐Aguilar, K.A. and Kuchta, R.D. 2004. Herpes simplex virus 1 DNA primase: A polymerase with extraordinarily low fidelity. Biochemistry 43:9084‐9091.
   Ramirez‐Aguilar, K.A., Moore, C.L., and Kuchta, R.D. 2005. Herpes simplex virus I primase employs Watson‐Crick hydrogen bonding to identify cognate NTPs. Biochemistry 44:15585‐15593.
   Reardon, J.E. 1989. Herpes simplex virus type 1 DNA polymerase. Mechanism of inhibition by acyclovir triphosphate. J. Biol. Chem. 264:7405‐7411.
   Reardon, J.E. 1990. Herpes simplex virus type I DNA polymerase: Mechanism‐based affinity chromatography. J. Biol. Chem. 265:7112.
   Segel, I.H. 1975. Enzyme Kinetics. John Wiley and Sons, New York.
   Showalter, A.K. and Tsai, M.‐D. 2002. A reexamination of the nucleotide incorporation fidelity of DNA polymerases. Biochemistry 41:10571‐10576.
   Sintim, H.O. and Kool, E.T. 2006. Remarkable sensitivity to DNA base shape in the DNA polymerase active site. Angew. Chem. Int. Ed. 45:1974‐1979.
   Tchesnokov, E.P., Obikhod, A., Schinazi, R.B., and Gotte, M. 2008. Delayed chain termination protects the anti‐hepatitis B virus drug entecavir from excision by HIV‐1 reverse transcriptase. J. Biol. Chem. 283:34218‐34228.
   Te Velthuis, A.J., Arnold, J.J., Cameron, C.E., van de Worm, S.J., and Snijder, E.J. 2010. The RNA polymerase activity of SARS‐coronavirus nsp12 is primer dependent. Nucleic Acids Res. 38:203‐214.
   Thompson, H. and Kuchta, R.D. 1995. Arabinofuranosyl nucleotides are not chain‐terminators during initiation of new strands of DNA by DNA polymerase alpha‐primase. Biochemistry 34:11198‐11203.
   Tsai, Y.‐C. and Johnson, K.A. 2006. A new paradigm for DNA polymerase specificity. Biochemistry 45:9675‐9687.
   Urban, M., Joubert, N., Hocek, M., Alexander, R.E., and Kuchta, R.D. 2009. Herpes simplex virus‐1 DNA primase: A remarkably inaccurate yet selective polymerase. Biochemistry 48:10866‐10881.
   Villarreal, E.C. 2001. Current and potential therapies for the treatment of herpes virus infections. Progr. Drug Res. 185‐228.
   Vivet‐Boudou, V., Didierjean, J., Isel, C., and Marquet, R. 2006. Nucleoside and nucleotide inhibitors of HIV‐1 replication. Cell. Mol. Life Sci. 63:163‐186.
   Wang, Q., Tullius, T.D., and Levin, J.R. 2007. Effects of discontinuities in the DNA template on abortive initiation and promoter escape by Escherichia coli RNA polymerase. J. Biol. Chem. 282:26917‐26927.
   Washington, M.T., Helquist, S.A., Kool, E.T., Prakash, L., and Prakash, S. 2003. Requirement of Watson‐Crick hydrogen bonding for DNA synthesis by yeast DNA polymerase eta. Mol. Cell. Biol. 23:5107‐5112.
   Wong, I., Patel, S.S., and Johnson, K.A. 1991. An induced fit kinetic mechanism for DNA replication fidelity: Direct measurement by single turnover kinetics. Biochemistry 30:526‐537.
   Wu, W., Bergstrom, D.E., and Jo Davisson, V. 2004. Chemoenzymatic preparation of nucleoside triphoshates. Curr. Protoc. Nucleic Acid Chem. 16:13.2.1‐13.2.19.
   Zhang, X., Lee, I., and Berdis, A.J. 2005. The use of nonnatural nucleotides to probe the contribution of shape complementarity and pi electron surface area during DNA polymerization. Biochemistry 44:13101‐13110.
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