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. 1998 Apr 1;331(Pt 1):79–87. doi: 10.1042/bj3310079

DNA polymerase beta: effects of gapped DNA substrates on dNTP specificity, fidelity, processivity and conformational changes.

J Ahn 1, V S Kraynov 1, X Zhong 1, B G Werneburg 1, M D Tsai 1
PMCID: PMC1219323  PMID: 9512464

Abstract

Pre-steady-state kinetic analysis was used to compare the catalytic properties of DNA polymerase beta (Pol beta) for single-base gap-filling and regular duplex DNA synthesis. The rate of polymerization (kpol) and the apparent equilibrium dissociation constant of dNTP (Kd) were determined with single-nucleotide gapped DNA substrates for all four possible correct base pairs and twelve possible incorrect base pairs, and the results were compared with those obtained previously with non-gapped primer/template duplex DNA substrates. For correct dNTP incorporation, the use of single-nucleotide gapped DNA led to significant decreases in the Kd of dNTP. Although kpol was little affected, the catalytic efficiency kpol/Kd increased significantly owing to the decreases in Kd. In contrast, for incorrect dNTP incorporation, the use of single-nucleotide gapped DNA substrates did not affect the Kd of dNTP appreciably but caused the kpol (and thus kpol/Kd) for incorrect dNTP incorporation to increase. As a consequence the fidelity of Pol beta was not significantly affected by the use of single-nucleotide gapped DNA substrates. In addition we show that under processive polymerization conditions the processivity of Pol beta increases in the gap-filling synthesis owing to a decreased rate of DNA dissociation. Finally, with a single-nucleotide gapped DNA substrate the rate-limiting conformational change step before chemistry was also observed. However, the preceding fast conformational change observed with duplex DNA substrates was not clearly detected. A possible cause is that in the complex with the gapped DNA, the 8 kDa N-terminal domain of Pol beta already exists in a closed conformation. This interpretation was supported by tryptic digestion experiments.

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Selected References

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  1. Ahn J., Werneburg B. G., Tsai M. D. DNA polymerase beta: structure-fidelity relationship from Pre-steady-state kinetic analyses of all possible correct and incorrect base pairs for wild type and R283A mutant. Biochemistry. 1997 Feb 4;36(5):1100–1107. doi: 10.1021/bi961653o. [DOI] [PubMed] [Google Scholar]
  2. Barshop B. A., Wrenn R. F., Frieden C. Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. Anal Biochem. 1983 Apr 1;130(1):134–145. doi: 10.1016/0003-2697(83)90660-7. [DOI] [PubMed] [Google Scholar]
  3. Caldecott K. W., Aoufouchi S., Johnson P., Shall S. XRCC1 polypeptide interacts with DNA polymerase beta and possibly poly (ADP-ribose) polymerase, and DNA ligase III is a novel molecular 'nick-sensor' in vitro. Nucleic Acids Res. 1996 Nov 15;24(22):4387–4394. doi: 10.1093/nar/24.22.4387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Casas-Finet J. R., Kumar A., Morris G., Wilson S. H., Karpel R. L. Spectroscopic studies of the structural domains of mammalian DNA beta-polymerase. J Biol Chem. 1991 Oct 15;266(29):19618–19625. [PubMed] [Google Scholar]
  5. Date T., Yamaguchi M., Hirose F., Nishimoto Y., Tanihara K., Matsukage A. Expression of active rat DNA polymerase beta in Escherichia coli. Biochemistry. 1988 Apr 19;27(8):2983–2990. doi: 10.1021/bi00408a048. [DOI] [PubMed] [Google Scholar]
  6. Dianov G., Price A., Lindahl T. Generation of single-nucleotide repair patches following excision of uracil residues from DNA. Mol Cell Biol. 1992 Apr;12(4):1605–1612. doi: 10.1128/mcb.12.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fishel R., Lescoe M. K., Rao M. R., Copeland N. G., Jenkins N. A., Garber J., Kane M., Kolodner R. The human mutator gene homolog MSH2 and its association with hereditary nonpolyposis colon cancer. Cell. 1993 Dec 3;75(5):1027–1038. doi: 10.1016/0092-8674(93)90546-3. [DOI] [PubMed] [Google Scholar]
  8. Holm L., Sander C. DNA polymerase beta belongs to an ancient nucleotidyltransferase superfamily. Trends Biochem Sci. 1995 Sep;20(9):345–347. doi: 10.1016/s0968-0004(00)89071-4. [DOI] [PubMed] [Google Scholar]
  9. Johnson K. A. Conformational coupling in DNA polymerase fidelity. Annu Rev Biochem. 1993;62:685–713. doi: 10.1146/annurev.bi.62.070193.003345. [DOI] [PubMed] [Google Scholar]
  10. Kati W. M., Johnson K. A., Jerva L. F., Anderson K. S. Mechanism and fidelity of HIV reverse transcriptase. J Biol Chem. 1992 Dec 25;267(36):25988–25997. [PubMed] [Google Scholar]
  11. Kraynov V. S., Werneburg B. G., Zhong X., Lee H., Ahn J., Tsai M. D. DNA polymerase beta: analysis of the contributions of tyrosine-271 and asparagine-279 to substrate specificity and fidelity of DNA replication by pre-steady-state kinetics. Biochem J. 1997 Apr 1;323(Pt 1):103–111. doi: 10.1042/bj3230103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kuchta R. D., Mizrahi V., Benkovic P. A., Johnson K. A., Benkovic S. J. Kinetic mechanism of DNA polymerase I (Klenow). Biochemistry. 1987 Dec 15;26(25):8410–8417. doi: 10.1021/bi00399a057. [DOI] [PubMed] [Google Scholar]
  13. Kunkel T. A., Alexander P. S. The base substitution fidelity of eucaryotic DNA polymerases. Mispairing frequencies, site preferences, insertion preferences, and base substitution by dislocation. J Biol Chem. 1986 Jan 5;261(1):160–166. [PubMed] [Google Scholar]
  14. Leach F. S., Nicolaides N. C., Papadopoulos N., Liu B., Jen J., Parsons R., Peltomäki P., Sistonen P., Aaltonen L. A., Nyström-Lahti M. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell. 1993 Dec 17;75(6):1215–1225. doi: 10.1016/0092-8674(93)90330-s. [DOI] [PubMed] [Google Scholar]
  15. Lehmann A. R. Nucleotide excision repair and the link with transcription. Trends Biochem Sci. 1995 Oct;20(10):402–405. doi: 10.1016/s0968-0004(00)89088-x. [DOI] [PubMed] [Google Scholar]
  16. Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. doi: 10.1038/362709a0. [DOI] [PubMed] [Google Scholar]
  17. Lindell T. J., Weinberg F., Morris P. W., Roeder R. G., Rutter W. J. Specific inhibition of nuclear RNA polymerase II by alpha-amanitin. Science. 1970 Oct 23;170(3956):447–449. doi: 10.1126/science.170.3956.447. [DOI] [PubMed] [Google Scholar]
  18. Matsukage A., Nishikawa K., Ooi T., Seto Y., Yamaguchi M. Homology between mammalian DNA polymerase beta and terminal deoxynucleotidyltransferase. J Biol Chem. 1987 Jul 5;262(19):8960–8962. [PubMed] [Google Scholar]
  19. Matsumoto Y., Kim K. Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science. 1995 Aug 4;269(5224):699–702. doi: 10.1126/science.7624801. [DOI] [PubMed] [Google Scholar]
  20. Modrich P. Mismatch repair, genetic stability, and cancer. Science. 1994 Dec 23;266(5193):1959–1960. doi: 10.1126/science.7801122. [DOI] [PubMed] [Google Scholar]
  21. Munn M. M., Alberts B. M. DNA footprinting studies of the complex formed by the T4 DNA polymerase holoenzyme at a primer-template junction. J Biol Chem. 1991 Oct 25;266(30):20034–20044. [PubMed] [Google Scholar]
  22. Munn M. M., Alberts B. M. The T4 DNA polymerase accessory proteins form an ATP-dependent complex on a primer-template junction. J Biol Chem. 1991 Oct 25;266(30):20024–20033. [PubMed] [Google Scholar]
  23. Narayan S., He F., Wilson S. H. Activation of the human DNA polymerase beta promoter by a DNA-alkylating agent through induced phosphorylation of cAMP response element-binding protein-1. J Biol Chem. 1996 Aug 2;271(31):18508–18513. doi: 10.1074/jbc.271.31.18508. [DOI] [PubMed] [Google Scholar]
  24. Parsons R., Li G. M., Longley M. J., Fang W. H., Papadopoulos N., Jen J., de la Chapelle A., Kinzler K. W., Vogelstein B., Modrich P. Hypermutability and mismatch repair deficiency in RER+ tumor cells. Cell. 1993 Dec 17;75(6):1227–1236. doi: 10.1016/0092-8674(93)90331-j. [DOI] [PubMed] [Google Scholar]
  25. Parsons R., Li G. M., Longley M., Modrich P., Liu B., Berk T., Hamilton S. R., Kinzler K. W., Vogelstein B. Mismatch repair deficiency in phenotypically normal human cells. Science. 1995 May 5;268(5211):738–740. doi: 10.1126/science.7632227. [DOI] [PubMed] [Google Scholar]
  26. Patel S. S., Wong I., Johnson K. A. Pre-steady-state kinetic analysis of processive DNA replication including complete characterization of an exonuclease-deficient mutant. Biochemistry. 1991 Jan 15;30(2):511–525. doi: 10.1021/bi00216a029. [DOI] [PubMed] [Google Scholar]
  27. Pelletier H., Sawaya M. R., Kumar A., Wilson S. H., Kraut J. Structures of ternary complexes of rat DNA polymerase beta, a DNA template-primer, and ddCTP. Science. 1994 Jun 24;264(5167):1891–1903. [PubMed] [Google Scholar]
  28. Prasad R., Singhal R. K., Srivastava D. K., Molina J. T., Tomkinson A. E., Wilson S. H. Specific interaction of DNA polymerase beta and DNA ligase I in a multiprotein base excision repair complex from bovine testis. J Biol Chem. 1996 Jul 5;271(27):16000–16007. doi: 10.1074/jbc.271.27.16000. [DOI] [PubMed] [Google Scholar]
  29. Sawaya M. R., Prasad R., Wilson S. H., Kraut J., Pelletier H. Crystal structures of human DNA polymerase beta complexed with gapped and nicked DNA: evidence for an induced fit mechanism. Biochemistry. 1997 Sep 16;36(37):11205–11215. doi: 10.1021/bi9703812. [DOI] [PubMed] [Google Scholar]
  30. Shinohara A., Ogawa T. Homologous recombination and the roles of double-strand breaks. Trends Biochem Sci. 1995 Oct;20(10):387–391. doi: 10.1016/s0968-0004(00)89085-4. [DOI] [PubMed] [Google Scholar]
  31. Singhal R. K., Prasad R., Wilson S. H. DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem. 1995 Jan 13;270(2):949–957. doi: 10.1074/jbc.270.2.949. [DOI] [PubMed] [Google Scholar]
  32. Singhal R. K., Wilson S. H. Short gap-filling synthesis by DNA polymerase beta is processive. J Biol Chem. 1993 Jul 25;268(21):15906–15911. [PubMed] [Google Scholar]
  33. Sobol R. W., Horton J. K., Kühn R., Gu H., Singhal R. K., Prasad R., Rajewsky K., Wilson S. H. Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature. 1996 Jan 11;379(6561):183–186. doi: 10.1038/379183a0. [DOI] [PubMed] [Google Scholar]
  34. Srivastava D. K., Rawson T. Y., Showalter S. D., Wilson S. H. Phorbol ester abrogates up-regulation of DNA polymerase beta by DNA-alkylating agents in Chinese hamster ovary cells. J Biol Chem. 1995 Jul 7;270(27):16402–16408. doi: 10.1074/jbc.270.27.16402. [DOI] [PubMed] [Google Scholar]
  35. Tabor S., Huber H. E., Richardson C. C. Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem. 1987 Nov 25;262(33):16212–16223. [PubMed] [Google Scholar]
  36. Tanabe K., Bohn E. W., Wilson S. H. Steady-state kinetics of mouse DNA polymerase beta. Biochemistry. 1979 Jul 24;18(15):3401–3406. doi: 10.1021/bi00582a029. [DOI] [PubMed] [Google Scholar]
  37. Wang T. S., Korn D. Specificity of the catalytic interaction of human DNA polymerase beta with nucleic acid substrates. Biochemistry. 1982 Mar 30;21(7):1597–1608. doi: 10.1021/bi00536a021. [DOI] [PubMed] [Google Scholar]
  38. Werneburg B. G., Ahn J., Zhong X., Hondal R. J., Kraynov V. S., Tsai M. D. DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity. Biochemistry. 1996 Jun 4;35(22):7041–7050. doi: 10.1021/bi9527202. [DOI] [PubMed] [Google Scholar]
  39. Wiebauer K., Jiricny J. Mismatch-specific thymine DNA glycosylase and DNA polymerase beta mediate the correction of G.T mispairs in nuclear extracts from human cells. Proc Natl Acad Sci U S A. 1990 Aug;87(15):5842–5845. doi: 10.1073/pnas.87.15.5842. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Young M. C., Reddy M. K., von Hippel P. H. Structure and function of the bacteriophage T4 DNA polymerase holoenzyme. Biochemistry. 1992 Sep 22;31(37):8675–8690. doi: 10.1021/bi00152a001. [DOI] [PubMed] [Google Scholar]
  41. Zhong X., Patel S. S., Werneburg B. G., Tsai M. D. DNA polymerase beta: multiple conformational changes in the mechanism of catalysis. Biochemistry. 1997 Sep 30;36(39):11891–11900. doi: 10.1021/bi963181j. [DOI] [PubMed] [Google Scholar]
  42. Zimmerle C. T., Frieden C. Analysis of progress curves by simulations generated by numerical integration. Biochem J. 1989 Mar 1;258(2):381–387. doi: 10.1042/bj2580381. [DOI] [PMC free article] [PubMed] [Google Scholar]

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