Discrimination against purine–pyrimidine mispairs in the polymerase active site of DNA polymerase I: A structural explanation
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We previously identified five derivatives of Klenow fragment DNA polymerase that have lower fidelity because of amino acid substitutions in the polymerase active site. One of these has alanine substituted for the invariant Glu-710, whose side chain interacts with the deoxyribose of the incoming dNTP. Here we show that the E710A enzyme has reduced fidelity for five of the 12 possible mismatches. All but one of these involve misinsertion of pyrimidines, including two transition mismatches A-dCTP and G-dTTP. In contrast, E710A polymerase error rates for the reciprocal C-dATP and T-dGTP transition mismatches were similar to those of the wild-type enzyme. The kinetics of formation of correct base pairs and transition mismatches by the wild-type and E710A polymerases, combined with information on the structure of the DNA polymerase active site and the asymmetry of wobble base pairs, provides a plausible explanation for the differential effects of the E710A mutation on fidelity. The data suggest that the Glu-710 side chain plays a pivotal role in excluding wobble base pairs between template pyrimidines and purine triphosphates by steric clash. Moreover, this same side chain enhances the stability of incoming correct dNTPs, such that loss of this interaction on removal of the side chain leads to lower selectivity against mismatches involving incoming pyrimidines.
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These base substitution errors may be minor forms resulting from wobble base pairing, tautomerization, ionization, or anti-syn rotations of bases.
“These may be minor forms resulting from wobble base pairing (e.g.Ref. 43), tautomerization, ionization, or anti-syn rotations of bases (33).”
From DNA Replication Fidelity (2004) · cites this paper as [43]
Cites
Bradford, M. M. (1976) · Analytical Biochemistry · doi:10.1016/0003-2697(76)90527-3 · Cited by 255,302 · see citing works
SWISS‐MODEL and the Swiss‐Pdb Viewer: An environment for comparative protein modeling
Guex, N., & Peitsch, M. (1997) · Electrophoresis · doi:10.1002/elps.1150181505 · Cited by 11,589 · see citing works
Crystal structure of a bacteriophage T7 DNA replication complex at 2.2 Å resolution
Doublié, S., Tabor, S., Long, A. M., Richardson, C. C., & Ellenberger, T. (1998) · Nature · doi:10.1038/34593 · Cited by 1,177 · see citing works
Hydrogen bonding and biological specificity analysed by protein engineering
Fersht, A. R., Shi, J., Knill-Jones, J. W., Lowe, D. M., Wilkinson, A. J., Blow, D. M., Brick, P., Carter, P. M., Waye, M. M. Y., & Winter, G. P. (1985) · Nature · doi:10.1038/314235a0 · Cited by 1,056 · see citing works
Kunkel, T. A., & Bebenek, K. (2000) · Annual Review of Biochemistry · doi:10.1146/annurev.biochem.69.1.497 · Cited by 1,003 · see citing works
Self-citationThe Y-Family of DNA Polymerases
Ohmori, H., Friedberg, E. C., Fuchs, R. P., Goodman, M. F., Hanaoka, F., Hinkle, D. C., Kunkel, T. A., Lawrence, C. W., Livneh, Z., Nohmi, T., et al. (2001) · Molecular Cell · doi:10.1016/s1097-2765(01)00278-7 · Cited by 882 · see citing works
Self-citationFIDELITY MECHANISMS IN DNA REPLICATION
Echols, H., & Goodman, M. F. (1991) · Annual Review of Biochemistry · doi:10.1146/annurev.bi.60.070191.002401 · Cited by 712 · see citing works
Li, Y., Korolev, S., & Waksman, G. (1998) · The EMBO Journal · doi:10.1093/emboj/17.24.7514 · Cited by 689 · see citing works
FUNCTION AND STRUCTURE RELATIONSHIPS IN DNA POLYMERASES
Joyce, C. M., & Steitz, T. A. (1994) · Annual Review of Biochemistry · doi:10.1146/annurev.bi.63.070194.004021 · Cited by 620 · see citing works
Self-citationVisualizing DNA replication in a catalytically active Bacillus DNA polymerase crystal
Kiefer, J. R., Mao, C., Braman, J. C., & Beese, L. S. (1998) · Nature · doi:10.1038/34693 · Cited by 518 · see citing works
Loeb, L. A., & Kunkel, T. A. (1982) · Annual Review of Biochemistry · doi:10.1146/annurev.bi.51.070182.002241 · Cited by 511 · see citing works
Self-citationBiochemical Basis of DNA Replication Fidelity
Goodman, M. F., Creighton, S., Bloom, L. B., Petruska, J., & Kunkel, T. A. (1993) · Critical Reviews in Biochemistry and Molecular Biology · doi:10.3109/10409239309086792 · Cited by 446 · see citing works
Self-citationBrautigam, C. A., & Steitz, T. A. (1998) · Current Opinion in Structural Biology · doi:10.1016/s0959-440x(98)80010-9 · Cited by 377 · see citing works
Genetic and Crystallographic Studies of the 3′,5′-Exonucleolytic Site of DNA Polymerase I
Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L. S., Friedman, J. M., Joyce, C. M., & Steitz, T. A. (1988) · Science · doi:10.1126/science.2832946 · Cited by 341 · see citing works
Self-citationAn open and closed case for all polymerases
Doublié, S., Sawaya, M. R., & Ellenberger, T. (1999) · Structure · doi:10.1016/s0969-2126(99)80017-3 · Cited by 314 · see citing works
Structure of an adenine˙cytosine base pair in DNA and its implications for mismatch repair
Hunter, W. N., Brown, T., Anand, N. N., & Kennard, O. (1986) · Nature · doi:10.1038/320552a0 · Cited by 281 · see citing works
Polesky, A. H., Steitz, T. A., Grindley, N. D. F., & Joyce, C. M. (1990) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)77342-0 · Cited by 259 · see citing works
Self-citationChoosing the right sugar: How polymerases select a nucleotide substrate
Joyce, C. M. (1997) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.94.5.1619 · Cited by 222 · see citing works
Self-citationThe many faces of DNA polymerases: Strategies for mutagenesis and for mutational avoidance
Friedberg, E. C., Feaver, W. J., & Gerlach, V. L. (2000) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.120152397 · Cited by 222 · see citing works
Astatke, M., Ng, K., Grindley, N. D. F., & Joyce, C. M. (1998) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.95.7.3402 · Cited by 212 · see citing works
Self-citation[18] Analyzing fidelity of DNA polymerases
Bębenek, K., & Kunkel, T. A. (1995) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/0076-6879(95)62020-6 · Cited by 198 · see citing works
Self-citationZhou, B.-L., Pata, J. D., & Steitz, T. A. (2001) · Molecular Cell · doi:10.1016/s1097-2765(01)00310-0 · Cited by 198 · see citing works
The fidelity of DNA synthesis catalyzed by derivatives of Escherichia coli DNA polymerase I.
Bębenek, K., Joyce, C. M., Fitzgerald, M. P., & Kunkel, T. A. (1990) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)77430-9 · Cited by 175 · see citing works
Self-citationPatel, D. J., Kozlowski, S. A., Marky, L. A., et al. (1982) · Biochemistry · doi:10.1021/bi00532a003 · Cited by 158 · see citing works
High-resolution structure of a DNA helix containing mismatched base pairs
Brown, T., Kennard, O., Kneale, G. G., & Rabinovich, D. (1985) · Nature · doi:10.1038/315604a0 · Cited by 132 · see citing works
Minnick, D. T., Bębenek, K., Osheroff, W. P., Turner, R. M., Astatke, M., Liu, L., Kunkel, T. A., & Joyce, C. M. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.5.3067 · Cited by 129 · see citing works
Self-citationLi, Y., Mitaxov, V., & Waksman, G. (1999) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.96.17.9491 · Cited by 127 · see citing works
A mutant of DNA polymerase I (Klenow fragment) with reduced fidelity
Carroll, S. S., Cowart, M., & Benkovic, S. J. (1991) · Biochemistry · doi:10.1021/bi00217a034 · Cited by 125 · see citing works
How E. coli DNA polymerase I (klenow fragment) distinguishes between deoxy- and dideoxynucleotides
Astatke, M., Grindley, N. D. F., & Joyce, C. M. (1998) · Journal of Molecular Biology · doi:10.1006/jmbi.1998.1672 · Cited by 109 · see citing works
Self-citationA Single Highly Mutable Catalytic Site Amino Acid Is Critical for DNA Polymerase Fidelity
Patel, P. H., Kawate, H., Adman, E. T., Ashbach, M. N., & Loeb, L. A. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m008701200 · Cited by 108 · see citing works
Sloppier copier DNA polymerases involved in genome repair
Goodman, M. F., & Tippin, B. L. (2000) · Current Opinion in Genetics & Development · doi:10.1016/s0959-437x(00)00057-5 · Cited by 107 · see citing works
Recent studies of the fidelity of DNA synthesis
Kunkel, T. A., & Bębenek, K. (1988) · Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression · doi:10.1016/0167-4781(88)90020-6 · Cited by 98 · see citing works
Self-citationLi, Y., & Waksman, G. (2001) · Protein Science · doi:10.1110/ps.250101 · Cited by 86 · see citing works
Bell, J. B., Eckert, K. A., Joyce, C. M., & Kunkel, T. A. (1997) · Journal of Biological Chemistry · doi:10.1074/jbc.272.11.7345 · Cited by 76 · see citing works
Self-citationJoyce, C. M., Sun, X. C., & Grindley, N. D. F. (1992) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)35792-2 · Cited by 74 · see citing works
Self-citationRefined crystal structure of an octanucleotide duplex with G · T mismatched base-pairs
Hunter, W. N., Kneale, G. G., Brown, T., Rabinovich, D., & Kennard, O. (1986) · Journal of Molecular Biology · doi:10.1016/0022-2836(86)90246-9 · Cited by 71 · see citing works
Base Substitution Specificity of DNA Polymerase β Depends on Interactions in the DNA Minor Groove
Osheroff, W. P., Beard, W. A., Wilson, S. H., & Kunkel, T. A. (1999) · Journal of Biological Chemistry · doi:10.1074/jbc.274.30.20749 · Cited by 57 · see citing works
Self-citation[1] Purification of Escherichia coli DNA polymerase I and Klenow fragment
Joyce, C. M., & Derbyshire, V. (1995) · Methods in enzymology on CD-ROM/Methods in enzymology · doi:10.1016/0076-6879(95)62003-6 · Cited by 54 · see citing works
Self-citationThe Conserved Active Site Motif A of Escherichia coliDNA Polymerase I Is Highly Mutable
Shinkai, A., Patel, P. H., & Loeb, L. A. (2001) · Journal of Biological Chemistry · doi:10.1074/jbc.m011472200 · Cited by 51 · see citing works
Gao, X., & Patel, D. J. (1987) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)45480-4 · Cited by 47 · see citing works
Base mismatches and mutagenesis: how important is tautomerism?
Morgan, A. R. (1993) · Trends in Biochemical Sciences · doi:10.1016/0968-0004(93)90104-u · Cited by 38 · see citing works
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Kunkel, T. A. (2004) · Journal of Biological Chemistry · doi:10.1074/jbc.r400006200 · Cited by 754 · see citing works
Self-citationKunkel, T. A., Pavlov, Y. I., & Bebenek, K. (2003) · DNA Repair · doi:10.1016/s1568-7864(02)00224-0 · Cited by 125 · see citing works
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- Authors
- Minnick, D. T., Liu, L., Grindley, N. D. F., Kunkel, T. A., & Joyce, C. M.
- Affiliations
- National Institutes of Health (US)
- Corresponding author
- Minnick, Dana T.
- Liu, Lixing
- Joyce, Catherine M.
- Venue
- Proceedings of the National Academy of Sciences
- Publisher
- National Academy of Sciences
- Published
- 2002-02-05
- Volume
- 99
- Issue
- 3
- Pages
- 1194-1199
- ISSN
- 0027-8424 (ISSN-L)
- 1091-6490
- PMC
- PMC122166
- OpenAlex
- W2089845127
- Cited by
- 37
- References
- 41
- Citations by year
- 2025: 1 · 2022: 1 · 2017: 3 · 2015: 1 · 2014: 1 · 2013: 3 · 2012: 2
- FWCI
- 0.93
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Proceedings of the National Academy of Sciences (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of General Medical Sciences (GM-28550, R01GM028550)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
- Physical Sciences › Materials Science › Materials Chemistry › Enzyme Structure and Function
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Keywords
- DNA polymerase I, Klenow fragment, mismatch discrimination, polymerase fidelity, wobble pairing, steric clashes, active site structure
- MeSH terms
- Binding Sites, Deoxyribonucleotides, DNA Polymerase I, Kinetics, Protein Conformation, Purines, Pyrimidines, Recombinant Proteins, Mutagenesis, Site-Directed, Amino Acid Substitution, Base Pair Mismatch
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