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. 1996 Jul 15;24(14):2774–2781. doi: 10.1093/nar/24.14.2774

Analysis and suppression of DNA polymerase pauses associated with a trinucleotide consensus.

D S Mytelka 1, M J Chamberlin 1
PMCID: PMC146000  PMID: 8759010

Abstract

We have studied a novel class of DNA sequences that cause DNA polymerases to pause. These sequences have the central consensus Py-G-C and are not necessarily adjacent to hairpins in the DNA template. Since most consensus sequences do not cause pauses under standard conditions, additional template features must exist that make it difficult to incorporate nucleotides at these positions. We believe that these pauses result from constraints that make the conformation change involved in nucleotide selection more difficult. These pauses can obscure parts of DNA sequencing ladders and prevent DNA amplification by the polymerase chain reaction. The addition of betaine, and some related compounds, relieves these pauses.

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Selected References

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  1. Abbotts J., Bebenek K., Kunkel T. A., Wilson S. H. Mechanism of HIV-1 reverse transcriptase. Termination of processive synthesis on a natural DNA template is influenced by the sequence of the template-primer stem. J Biol Chem. 1993 May 15;268(14):10312–10323. [PubMed] [Google Scholar]
  2. Arakawa T., Timasheff S. N. Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch Biochem Biophys. 1983 Jul 1;224(1):169–177. doi: 10.1016/0003-9861(83)90201-1. [DOI] [PubMed] [Google Scholar]
  3. Barnes W. M. PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2216–2220. doi: 10.1073/pnas.91.6.2216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bedinger P., Munn M., Alberts B. M. Sequence-specific pausing during in vitro DNA replication on double-stranded DNA templates. J Biol Chem. 1989 Oct 5;264(28):16880–16886. [PubMed] [Google Scholar]
  5. Boyer P. L., Tantillo C., Jacobo-Molina A., Nanni R. G., Ding J., Arnold E., Hughes S. H. Sensitivity of wild-type human immunodeficiency virus type 1 reverse transcriptase to dideoxynucleotides depends on template length; the sensitivity of drug-resistant mutants does not. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4882–4886. doi: 10.1073/pnas.91.11.4882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buche A., Colson P., Houssier C. Effect of organic effectors on chromatin solubility, DNA-histone H1 interactions, DNA and histone H1 structures. J Biomol Struct Dyn. 1993 Aug;11(1):95–119. doi: 10.1080/07391102.1993.10508712. [DOI] [PubMed] [Google Scholar]
  7. Challberg M. D., Englund P. T. The effect of template secondary structure on vaccinia DNA polymerase. J Biol Chem. 1979 Aug 25;254(16):7820–7826. [PubMed] [Google Scholar]
  8. Chuprina V. P., Heinemann U., Nurislamov A. A., Zielenkiewicz P., Dickerson R. E., Saenger W. Molecular dynamics simulation of the hydration shell of a B-DNA decamer reveals two main types of minor-groove hydration depending on groove width. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):593–597. doi: 10.1073/pnas.88.2.593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Del Sal G., Manfioletti G., Schneider C. The CTAB-DNA precipitation method: a common mini-scale preparation of template DNA from phagemids, phages or plasmids suitable for sequencing. Biotechniques. 1989 May;7(5):514–520. [PubMed] [Google Scholar]
  10. Drew H. R., Dickerson R. E. Structure of a B-DNA dodecamer. III. Geometry of hydration. J Mol Biol. 1981 Sep 25;151(3):535–556. doi: 10.1016/0022-2836(81)90009-7. [DOI] [PubMed] [Google Scholar]
  11. Fry M., Loeb L. A. A DNA polymerase alpha pause site is a hot spot for nucleotide misinsertion. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):763–767. doi: 10.1073/pnas.89.2.763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gotley D. C., Ball D. E., Owen R. W., Williamson R. C., Cooper M. J. Evaluation and surgical correction of esophagitis after partial gastrectomy. Surgery. 1992 Jan;111(1):29–36. [PubMed] [Google Scholar]
  13. Hacker K. J., Alberts B. M. The rapid dissociation of the T4 DNA polymerase holoenzyme when stopped by a DNA hairpin helix. A model for polymerase release following the termination of each Okazaki fragment. J Biol Chem. 1994 Sep 30;269(39):24221–24228. [PubMed] [Google Scholar]
  14. Hogan M., LeGrange J., Austin B. Dependence of DNA helix flexibility on base composition. Nature. 1983 Aug 25;304(5928):752–754. doi: 10.1038/304752a0. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. LaDuca R. J., Fay P. J., Chuang C., McHenry C. S., Bambara R. A. Site-specific pausing of deoxyribonucleic acid synthesis catalyzed by four forms of Escherichia coli DNA polymerase III. Biochemistry. 1983 Oct 25;22(22):5177–5188. doi: 10.1021/bi00291a018. [DOI] [PubMed] [Google Scholar]
  17. Levin J. R., Chamberlin M. J. Mapping and characterization of transcriptional pause sites in the early genetic region of bacteriophage T7. J Mol Biol. 1987 Jul 5;196(1):61–84. doi: 10.1016/0022-2836(87)90511-0. [DOI] [PubMed] [Google Scholar]
  18. Lin T. Y., Timasheff S. N. Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein. Biochemistry. 1994 Oct 25;33(42):12695–12701. doi: 10.1021/bi00208a021. [DOI] [PubMed] [Google Scholar]
  19. Melchior W. B., Jr, Von Hippel P. H. Alteration of the relative stability of dA-dT and dG-dC base pairs in DNA. Proc Natl Acad Sci U S A. 1973 Feb;70(2):298–302. doi: 10.1073/pnas.70.2.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myers T. W., Romano L. J. Mechanism of stimulation of T7 DNA polymerase by Escherichia coli single-stranded DNA binding protein (SSB). J Biol Chem. 1988 Nov 15;263(32):17006–17015. [PubMed] [Google Scholar]
  21. Reckmann B., Grosse F., Urbanke C., Frank R., Blöcker H., Krauss G. Analysis of secondary structures in M13mp8 (+) single-stranded DNA by the pausing of DNA polymerase alpha. Eur J Biochem. 1985 Nov 4;152(3):633–643. doi: 10.1111/j.1432-1033.1985.tb09242.x. [DOI] [PubMed] [Google Scholar]
  22. Rees W. A., Yager T. D., Korte J., von Hippel P. H. Betaine can eliminate the base pair composition dependence of DNA melting. Biochemistry. 1993 Jan 12;32(1):137–144. doi: 10.1021/bi00052a019. [DOI] [PubMed] [Google Scholar]
  23. Russel M., Kidd S., Kelley M. R. An improved filamentous helper phage for generating single-stranded plasmid DNA. Gene. 1986;45(3):333–338. doi: 10.1016/0378-1119(86)90032-6. [DOI] [PubMed] [Google Scholar]
  24. Santoro M. M., Liu Y., Khan S. M., Hou L. X., Bolen D. W. Increased thermal stability of proteins in the presence of naturally occurring osmolytes. Biochemistry. 1992 Jun 16;31(23):5278–5283. doi: 10.1021/bi00138a006. [DOI] [PubMed] [Google Scholar]
  25. Shapiro J. T., Leng M., Felsenfeld G. Deoxyribonucleic acid-polylysine complexes. Structure and nucleotide specificity. Biochemistry. 1969 Aug;8(8):3219–3232. doi: 10.1021/bi00836a014. [DOI] [PubMed] [Google Scholar]
  26. Sherman L. A., Gefter M. L. Studies on the mechanism of enzymatic DNA elongation by Escherichia coli DNA polymerase II. J Mol Biol. 1976 May 5;103(1):61–76. doi: 10.1016/0022-2836(76)90052-8. [DOI] [PubMed] [Google Scholar]
  27. Suzuki M., Izuta S., Yoshida S. DNA polymerase alpha overcomes an error-prone pause site in the presence of replication protein-A. J Biol Chem. 1994 Apr 8;269(14):10225–10228. [PubMed] [Google Scholar]
  28. 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]
  29. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tabor S., Richardson C. C. Selective inactivation of the exonuclease activity of bacteriophage T7 DNA polymerase by in vitro mutagenesis. J Biol Chem. 1989 Apr 15;264(11):6447–6458. [PubMed] [Google Scholar]
  31. Thakar M., Bilenko A., Becktel W. J. Osmolyte mediation of T7 DNA polymerase and plasmid DNA stability. Biochemistry. 1994 Oct 11;33(40):12255–12259. doi: 10.1021/bi00206a031. [DOI] [PubMed] [Google Scholar]
  32. Weaver D. T., DePamphilis M. L. Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha. J Biol Chem. 1982 Feb 25;257(4):2075–2086. [PubMed] [Google Scholar]
  33. Weaver D. T., DePamphilis M. L. The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in vivo. J Mol Biol. 1984 Dec 25;180(4):961–986. doi: 10.1016/0022-2836(84)90266-3. [DOI] [PubMed] [Google Scholar]
  34. Winzor C. L., Winzor D. J., Paleg L. G., Jones G. P., Naidu B. P. Rationalization of the effects of compatible solutes on protein stability in terms of thermodynamic nonideality. Arch Biochem Biophys. 1992 Jul;296(1):102–107. doi: 10.1016/0003-9861(92)90550-g. [DOI] [PubMed] [Google Scholar]
  35. Yancey P. H., Clark M. E., Hand S. C., Bowlus R. D., Somero G. N. Living with water stress: evolution of osmolyte systems. Science. 1982 Sep 24;217(4566):1214–1222. doi: 10.1126/science.7112124. [DOI] [PubMed] [Google Scholar]

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