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. 1990 Aug 11;18(15):4543–4552. doi: 10.1093/nar/18.15.4543

Effect of monovalent cation-induced telomeric DNA structure on the binding of Oxytricha telomeric protein.

M K Raghuraman 1, T R Cech 1
PMCID: PMC331275  PMID: 2388834

Abstract

Oligonucleotides bearing 4 repeats of telomeric deoxyguanosine-rich sequence undergo a monovalent cation-induced transition to a folded conformation with G-G base pairs, modeled as a 'G-quartet' structure. We have now deduced the rates of folding and unfolding of d(TTTTGGGG)4, which has four repeats of the Oxytricha telomeric DNA sequence. The estimated average values of delta G for the folded form at 37 degrees C are -2.2 kcal/mol and -4.7 kcal/mol in 50 mM na+ and K+, respectively. The fully folded DNA is not recognized by the Oxytricha telomere-binding protein; the substrate for protein binding has properties consistent with its being partly or fully unfolded. In confirmation of this conclusion, prevention of DNA folding by methylation enables the protein to bind as rapidly in the presence of monovalent cations as in their absence. The slow unfolding (t1/2 = 4 hr and 18 hr at 37 degrees C in Na+ and K+, respectively) of the DNA suggests that such structures would be long-lived if they formed in vivo, unless they can be actively unfolded. The inability of the telomere-binding protein to bind the stable, folded form of the 4-repeat telomeric sequence is a problem that may be circumvented in vivo by avoiding four single-stranded repeats.

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Selected References

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  1. Agarwal K. L., Brunstedt J., Noyes B. E. A general method for detection and characterization of an mRNA using an oligonucleotide probe. J Biol Chem. 1981 Jan 25;256(2):1023–1028. [PubMed] [Google Scholar]
  2. Blackburn E. H., Gall J. G. A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena. J Mol Biol. 1978 Mar 25;120(1):33–53. doi: 10.1016/0022-2836(78)90294-2. [DOI] [PubMed] [Google Scholar]
  3. Blackburn E. H. The molecular structure of centromeres and telomeres. Annu Rev Biochem. 1984;53:163–194. doi: 10.1146/annurev.bi.53.070184.001115. [DOI] [PubMed] [Google Scholar]
  4. Britten R. J., Graham D. E., Neufeld B. R. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 1974;29:363–418. doi: 10.1016/0076-6879(74)29033-5. [DOI] [PubMed] [Google Scholar]
  5. Cech T. R. G-strings at chromosome ends. Nature. 1988 Apr 28;332(6167):777–778. doi: 10.1038/332777a0. [DOI] [PubMed] [Google Scholar]
  6. GELLERT M., LIPSETT M. N., DAVIES D. R. Helix formation by guanylic acid. Proc Natl Acad Sci U S A. 1962 Dec 15;48:2013–2018. doi: 10.1073/pnas.48.12.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gamper H. B., Cimino G. D., Hearst J. E. Solution hybridization of crosslinkable DNA oligonucleotides to bacteriophage M13 DNA. Effect of secondary structure on hybridization kinetics and equilibria. J Mol Biol. 1987 Sep 20;197(2):349–362. doi: 10.1016/0022-2836(87)90128-8. [DOI] [PubMed] [Google Scholar]
  8. Gottschling D. E., Cech T. R. Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex. Cell. 1984 Sep;38(2):501–510. doi: 10.1016/0092-8674(84)90505-1. [DOI] [PubMed] [Google Scholar]
  9. Gottschling D. E., Zakian V. A. Telomere proteins: specific recognition and protection of the natural termini of Oxytricha macronuclear DNA. Cell. 1986 Oct 24;47(2):195–205. doi: 10.1016/0092-8674(86)90442-3. [DOI] [PubMed] [Google Scholar]
  10. Greider C. W., Blackburn E. H. A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature. 1989 Jan 26;337(6205):331–337. doi: 10.1038/337331a0. [DOI] [PubMed] [Google Scholar]
  11. Greider C. W., Blackburn E. H. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell. 1985 Dec;43(2 Pt 1):405–413. doi: 10.1016/0092-8674(85)90170-9. [DOI] [PubMed] [Google Scholar]
  12. Greider C. W., Blackburn E. H. The telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell. 1987 Dec 24;51(6):887–898. doi: 10.1016/0092-8674(87)90576-9. [DOI] [PubMed] [Google Scholar]
  13. Gruenwedel D. W., Hsu C. H., Lu D. S. The effects of aqueous neutral-salt solutions on the melting temperatures of deoxyribonucleic acids. Biopolymers. 1971;10(1):47–68. doi: 10.1002/bip.360100106. [DOI] [PubMed] [Google Scholar]
  14. Henderson E. R., Blackburn E. H. An overhanging 3' terminus is a conserved feature of telomeres. Mol Cell Biol. 1989 Jan;9(1):345–348. doi: 10.1128/mcb.9.1.345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henderson E. R., Moore M., Malcolm B. A. Telomere G-strand structure and function analyzed by chemical protection, base analogue substitution, and utilization by telomerase in vitro. Biochemistry. 1990 Jan 23;29(3):732–737. doi: 10.1021/bi00455a020. [DOI] [PubMed] [Google Scholar]
  16. Henderson E., Hardin C. C., Walk S. K., Tinoco I., Jr, Blackburn E. H. Telomeric DNA oligonucleotides form novel intramolecular structures containing guanine-guanine base pairs. Cell. 1987 Dec 24;51(6):899–908. doi: 10.1016/0092-8674(87)90577-0. [DOI] [PubMed] [Google Scholar]
  17. Klobutcher L. A., Swanton M. T., Donini P., Prescott D. M. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A. 1981 May;78(5):3015–3019. doi: 10.1073/pnas.78.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lipps H. J., Gruissem W., Prescott D. M. Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2495–2499. doi: 10.1073/pnas.79.8.2495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Marmur J., Lane D. STRAND SEPARATION AND SPECIFIC RECOMBINATION IN DEOXYRIBONUCLEIC ACIDS: BIOLOGICAL STUDIES. Proc Natl Acad Sci U S A. 1960 Apr;46(4):453–461. doi: 10.1073/pnas.46.4.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morin G. B. The human telomere terminal transferase enzyme is a ribonucleoprotein that synthesizes TTAGGG repeats. Cell. 1989 Nov 3;59(3):521–529. doi: 10.1016/0092-8674(89)90035-4. [DOI] [PubMed] [Google Scholar]
  21. Moyzis R. K., Buckingham J. M., Cram L. S., Dani M., Deaven L. L., Jones M. D., Meyne J., Ratliff R. L., Wu J. R. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6622–6626. doi: 10.1073/pnas.85.18.6622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oka Y., Shiota S., Nakai S., Nishida Y., Okubo S. Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata. Gene. 1980 Sep;10(4):301–306. doi: 10.1016/0378-1119(80)90150-x. [DOI] [PubMed] [Google Scholar]
  23. Oka Y., Thomas C. A., Jr The cohering telomeres of Oxytricha. Nucleic Acids Res. 1987 Nov 11;15(21):8877–8898. doi: 10.1093/nar/15.21.8877. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pluta A. F., Dani G. M., Spear B. B., Zakian V. A. Elaboration of telomeres in yeast: recognition and modification of termini from Oxytricha macronuclear DNA. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1475–1479. doi: 10.1073/pnas.81.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Price C. M., Cech T. R. Properties of the telomeric DNA-binding protein from Oxytricha nova. Biochemistry. 1989 Jan 24;28(2):769–774. doi: 10.1021/bi00428a053. [DOI] [PubMed] [Google Scholar]
  26. Price C. M., Cech T. R. Telomeric DNA-protein interactions of Oxytricha macronuclear DNA. Genes Dev. 1987 Oct;1(8):783–793. doi: 10.1101/gad.1.8.783. [DOI] [PubMed] [Google Scholar]
  27. Price C. M. Telomere structure in Euplotes crassus: characterization of DNA-protein interactions and isolation of a telomere-binding protein. Mol Cell Biol. 1990 Jul;10(7):3421–3431. doi: 10.1128/mcb.10.7.3421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Raghuraman M. K., Cech T. R. Assembly and self-association of oxytricha telomeric nucleoprotein complexes. Cell. 1989 Nov 17;59(4):719–728. doi: 10.1016/0092-8674(89)90018-4. [DOI] [PubMed] [Google Scholar]
  29. Raghuraman M. K., Dunn C. J., Hicke B. J., Cech T. R. Oxytricha telomeric nucleoprotein complexes reconstituted with synthetic DNA. Nucleic Acids Res. 1989 Jun 12;17(11):4235–4253. doi: 10.1093/nar/17.11.4235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Richards E. J., Ausubel F. M. Isolation of a higher eukaryotic telomere from Arabidopsis thaliana. Cell. 1988 Apr 8;53(1):127–136. doi: 10.1016/0092-8674(88)90494-1. [DOI] [PubMed] [Google Scholar]
  31. Sen D., Gilbert W. A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature. 1990 Mar 29;344(6265):410–414. doi: 10.1038/344410a0. [DOI] [PubMed] [Google Scholar]
  32. Sen D., Gilbert W. Formation of parallel four-stranded complexes by guanine-rich motifs in DNA and its implications for meiosis. Nature. 1988 Jul 28;334(6180):364–366. doi: 10.1038/334364a0. [DOI] [PubMed] [Google Scholar]
  33. Shampay J., Szostak J. W., Blackburn E. H. DNA sequences of telomeres maintained in yeast. Nature. 1984 Jul 12;310(5973):154–157. doi: 10.1038/310154a0. [DOI] [PubMed] [Google Scholar]
  34. Shippen-Lentz D., Blackburn E. H. Functional evidence for an RNA template in telomerase. Science. 1990 Feb 2;247(4942):546–552. doi: 10.1126/science.1689074. [DOI] [PubMed] [Google Scholar]
  35. Shippen-Lentz D., Blackburn E. H. Telomere terminal transferase activity from Euplotes crassus adds large numbers of TTTTGGGG repeats onto telomeric primers. Mol Cell Biol. 1989 Jun;9(6):2761–2764. doi: 10.1128/mcb.9.6.2761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sundquist W. I., Klug A. Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops. Nature. 1989 Dec 14;342(6251):825–829. doi: 10.1038/342825a0. [DOI] [PubMed] [Google Scholar]
  37. Swanton M. T., Greslin A. F., Prescott D. M. Arrangement of coding and non-coding sequences in the DNA molecules coding for rRNAs in Oxytricha sp. DNA of ciliated protozoa. VII. Chromosoma. 1980;77(2):203–215. doi: 10.1007/BF00329545. [DOI] [PubMed] [Google Scholar]
  38. Szostak J. W., Blackburn E. H. Cloning yeast telomeres on linear plasmid vectors. Cell. 1982 May;29(1):245–255. doi: 10.1016/0092-8674(82)90109-x. [DOI] [PubMed] [Google Scholar]
  39. Van Ness J., Hahn W. E. Physical parameters affecting the rate and completion of RNA driven hybridization of DNA: new measurements relevant to quantitation based on kinetics. Nucleic Acids Res. 1982 Dec 20;10(24):8061–8077. doi: 10.1093/nar/10.24.8061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wetmur J. G., Davidson N. Kinetics of renaturation of DNA. J Mol Biol. 1968 Feb 14;31(3):349–370. doi: 10.1016/0022-2836(68)90414-2. [DOI] [PubMed] [Google Scholar]
  41. Williamson J. R., Raghuraman M. K., Cech T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell. 1989 Dec 1;59(5):871–880. doi: 10.1016/0092-8674(89)90610-7. [DOI] [PubMed] [Google Scholar]
  42. Wu R. Nucleotide sequence analysis of DNA. Nat New Biol. 1972 Apr 19;236(68):198–200. doi: 10.1038/newbio236198a0. [DOI] [PubMed] [Google Scholar]
  43. Yu G. L., Bradley J. D., Attardi L. D., Blackburn E. H. In vivo alteration of telomere sequences and senescence caused by mutated Tetrahymena telomerase RNAs. Nature. 1990 Mar 8;344(6262):126–132. doi: 10.1038/344126a0. [DOI] [PubMed] [Google Scholar]
  44. Zahler A. M., Prescott D. M. DNA primase and the replication of the telomeres in Oxytricha nova. Nucleic Acids Res. 1989 Aug 11;17(15):6299–6317. doi: 10.1093/nar/17.15.6299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zahler A. M., Prescott D. M. Telomere terminal transferase activity in the hypotrichous ciliate Oxytricha nova and a model for replication of the ends of linear DNA molecules. Nucleic Acids Res. 1988 Jul 25;16(14B):6953–6972. doi: 10.1093/nar/16.14.6953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zimmerman S. B., Cohen G. H., Davies D. R. X-ray fiber diffraction and model-building study of polyguanylic acid and polyinosinic acid. J Mol Biol. 1975 Feb 25;92(2):181–192. doi: 10.1016/0022-2836(75)90222-3. [DOI] [PubMed] [Google Scholar]

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