Summary
The fluorescence of the base analog 2-aminopurine (2AP) is used in highly sensitive assays to determine kinetic parameters for DNA polymerase catalyzed reactions, including exonucleolytic proofreading and nucleotide binding and incorporation. Since 2AP fluorescence can also be used to probe DNA polymerase-induced conformational changes in 2AP-labeled DNA substrates, reaction steps that occur before product formation can be detected. Instruction is provided here in the use of 2AP fluorescence in steady-state and presteady-state assays to study DNA polymerase function and DNA replication.
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References
Ward, D.C., Reich, E., and Stryer, L. (1969) Fluorescence studies of nucleotides and polynucleotides. I. Formycin, 2-aminopurine riboside, 2,6-diaminopurine riboside, and their derivatives. J. Biol. Chem. 244, 1228–1237.
Rachofsky, E.L., Osman, R., and Ross, J.B.A. (2001) Probing structure and dynamics of DNA with 2-aminopurine: effects of local environment on fluorescence. Biochemistry 40, 946–956.
Bloom, L.B., Otto, M.R., Eritja, R., Reha-Krantz, L.J., Goodman, M.F. Beechem, J.M. (1994) Pre-steady-state kinetic analysis of sequence-dependent nucleotide excision by the 3′-exonuclease activity of bacteriophage T4 DNA polymerase. Biochemistry 33, 7576–7685.
Bloom, L.B., Otto, M.R., Beechem, J.M., and Goodman, M.F. (1993) Influence of 5′-nearest neighbors on the insertion kinetics of the fluorescence nucleotide analog 2-aminopurine by Klenow fragment. Biochemistry 32, 11247–11258.
Hariharan, C., Bloom, L.J., Helquist, S.A., Kool, E.T., and Reha-Krantz, L.J. (2006) Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies. Biochemistry 45, 2836–2844.
Sowers, L.C., Fazakerley, G.V., Eritja, R., Kaplan, B.E., and Goodman, M.F. (1986) Base pairing and mutagenesis: observation of a protonated base pair between 2-aminopurine and cytosine in an oligonucleotide by proton NMR. Proc. Natl. Acad. Sci. U. S. A. 83, 5434–5438.
Pless, R.C., Levitt, L.M. and Bessman, M.J. (1981) Nonrandom substitution of 2-aminopurine for adenine during deoxyribonucleic acid synthesis in vitro. Biochemistry 20, 6235–6244.
Frey, M.A., Sowers, L.C., Millar, D.P., and Benkovic, S.J. (1995) The nucleotide analog 2-aminopurine as a spectroscopic probe of nucleotide incorporation by the Klenow fragment of Escherichia coli polymerase I and bacteriophage T4 DNA polymerase. Biochemistry 34, 9185–9192.
Mandal, S.S., Fidalgo da Silva, E., and Reha-Krantz, L.J. (2002) Using 2-aminopurine fluorescence to detect base unstacking in the template strand during nucleotide incorporation by the bacteriophage T4 DNA polymerase. Biochemistry 41, 4399–4406.
Fidalgo da Silva, E., Mandal, S.S., and Reha-Krantz, L.J. (2002) Using 2-aminopurine fluorescence to measure incorporation of incorrect nucleotides by wild type and mutant bacteriophage T4 DNA polymerases. J. Biol. Chem. 277, 40640–40649.
Bessman, M.J., and Reha-Krantz, L.J. (1977) Studies on the biochemical basis of spontaneous mutation. V. Effect of temperature on mutation frequency. J. Mol. Biol. 116, 115–123.
Purohit, V., Grindley, N.D.F., and Joyce, C.M. (2003) Use of 2-aminopurine fluorescence to examine conformational changes during nucleotide incorporation by DNA polymerase I (Klenow fragment). Biochemistry 42, 10200–10211.
Dunlap, C.A., and Tsai, M-D. (2002) Use of 2-aminopurine and tryptophan fluorescence as probes in kinetic analyses of DNA polymerase β. Biochemistry 41, 11226–11235.
Tleugabulova, D., and Reha-Krantz, L.J. (2007) Probing DNA polymerase-DNA interactions: examining the template strand in exonuclease complexes using 2-aminopurin fluorescence and acrylamide quenching. Biochemistry 46, 6559–6569.
Hariharan, C., and Reha-Krantz, L.J. (2005) Using 2-aminopurine fluorescence to detect bacteriophage T4 DNA polymerase–DNA complexes that are important for primer extension and proofreading reactions. Biochemisty 44, 15674–15684.
Lakowicz, J.R. (1999) Principles of Fluorescence Spectroscopy. Plenum, New York.
Marquez, L.A., and Reha-Krantz, L.J. (1996) Using 2-aminopurine fluorescence and mutational analysis to demonstrate an active role of bacteriophage T4 DNA polymerase in strand separation required for 3′→5′ exonuclease activity. J. Biol. Chem. 271, 28903–28911.
Reddy, M.K., Weitzel, S.E., and von Hippel, P.H. (1992) Processive proofreading is intrinsic to T4 DNA polymerase. J. Biol. Chem. 267, 14157–14166.
Fidalgo da Silva, E., and Reha-Krantz, L.J. (2007) DNA polymerase proofreading: active site switching catalyzed by the bacteriophage T4 DNA polymerase. Nucleic Acids Res. 35, 5452–5463.
Reha-Krantz, L.J., Nonay, R.L., and Stocki, S. (1993) Bacteriophage T4 DNA polymerase mutations that confer sensitivity to the PPi analog phosphonoacetic acid. J. Virol. 67, 60–66.
Baker, R.P., and Reha-Krantz, L.J. (1998) Identification of a transient excision intermediate at the crossroads between DNA polymerase extension and proofreading pathway. Proc. Natl. Acad. Sci. U. S. A. 95, 3507–3512.
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Reha-Krantz, L.J. (2009). The Use of 2-Aminopurine Fluorescence to Study DNA Polymerase Function. In: Vengrova, S., Dalgaard, J. (eds) DNA Replication. Methods in Molecular Biology, vol 521. Humana Press. https://doi.org/10.1007/978-1-60327-815-7_21
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DOI: https://doi.org/10.1007/978-1-60327-815-7_21
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