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. 1998 Apr 1;26(7):1613–1620. doi: 10.1093/nar/26.7.1613

Purification, characterization and molecular cloning of TGP1, a novel G-DNA binding protein from Tetrahymena thermophila.

Q Lu 1, T Schierer 1, S G Kang 1, E Henderson 1
PMCID: PMC147456  PMID: 9512530

Abstract

G-DNA, a polymorphic family of four-stranded DNA structures, has been proposed to play roles in a variety of biological processes including telomere function, meiotic recombination and gene regulation. Here we report the purification and cloning of TGP1, a G-DNA specific binding protein from Tetrahymena thermophila. TGP1 was purified by three-column chromatographies, including a G-DNA affinity column. Two major proteins (approximately 80 and approximately 40 kDa) were present in the most highly purified column fraction. Renaturation experiments showed that the approximately 80 kDa protein contains TGP1 activity. Biochemical characterization showed that TGP1 is a G-DNA specific binding protein with a preference for parallel G-DNAs. The TGP1/DNA complex has a dissociation constant (Kd) of approximately 2.2 x 10(-8) M and TGP1 can form supershift in gel mobility shift assays. The cDNA coding TGP1 was cloned and sequenced based upon an internal peptide sequence obtained from the approximately 80 kDa protein. Sequence analyses showed that TGP1 is a basic protein with a pI of 10.58, and contains two extensively hydrophilic and basic domains. Homology searches revealed that TGP1 is a novel protein sharing weak similarities with a number of proteins.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Awang G., Sen D. Mode of dimerization of HIV-1 genomic RNA. Biochemistry. 1993 Oct 26;32(42):11453–11457. doi: 10.1021/bi00093a024. [DOI] [PubMed] [Google Scholar]
  3. Bashkirov V. I., Scherthan H., Solinger J. A., Buerstedde J. M., Heyer W. D. A mouse cytoplasmic exoribonuclease (mXRN1p) with preference for G4 tetraplex substrates. J Cell Biol. 1997 Feb 24;136(4):761–773. doi: 10.1083/jcb.136.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bilaud T., Koering C. E., Binet-Brasselet E., Ancelin K., Pollice A., Gasser S. M., Gilson E. The telobox, a Myb-related telomeric DNA binding motif found in proteins from yeast, plants and human. Nucleic Acids Res. 1996 Apr 1;24(7):1294–1303. doi: 10.1093/nar/24.7.1294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chung I. K., Mehta V. B., Spitzner J. R., Muller M. T. Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes. Nucleic Acids Res. 1992 Apr 25;20(8):1973–1977. doi: 10.1093/nar/20.8.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cooper J. P., Nimmo E. R., Allshire R. C., Cech T. R. Regulation of telomere length and function by a Myb-domain protein in fission yeast. Nature. 1997 Feb 20;385(6618):744–747. doi: 10.1038/385744a0. [DOI] [PubMed] [Google Scholar]
  7. Dionne I., Wellinger R. J. Cell cycle-regulated generation of single-stranded G-rich DNA in the absence of telomerase. Proc Natl Acad Sci U S A. 1996 Nov 26;93(24):13902–13907. doi: 10.1073/pnas.93.24.13902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Evans T., Schon E., Gora-Maslak G., Patterson J., Efstratiadis A. S1-hypersensitive sites in eukaryotic promoter regions. Nucleic Acids Res. 1984 Nov 12;12(21):8043–8058. doi: 10.1093/nar/12.21.8043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fang G., Cech T. R. Characterization of a G-quartet formation reaction promoted by the beta-subunit of the Oxytricha telomere-binding protein. Biochemistry. 1993 Nov 2;32(43):11646–11657. doi: 10.1021/bi00094a022. [DOI] [PubMed] [Google Scholar]
  10. Fang G., Cech T. R. The beta subunit of Oxytricha telomere-binding protein promotes G-quartet formation by telomeric DNA. Cell. 1993 Sep 10;74(5):875–885. doi: 10.1016/0092-8674(93)90467-5. [DOI] [PubMed] [Google Scholar]
  11. Frantz J. D., Gilbert W. A novel yeast gene product, G4p1, with a specific affinity for quadruplex nucleic acids. J Biol Chem. 1995 Sep 1;270(35):20692–20697. doi: 10.1074/jbc.270.35.20692. [DOI] [PubMed] [Google Scholar]
  12. Frantz J. D., Gilbert W. A yeast gene product, G4p2, with a specific affinity for quadruplex nucleic acids. J Biol Chem. 1995 Apr 21;270(16):9413–9419. doi: 10.1074/jbc.270.16.9413. [DOI] [PubMed] [Google Scholar]
  13. Fry M., Loeb L. A. The fragile X syndrome d(CGG)n nucleotide repeats form a stable tetrahelical structure. Proc Natl Acad Sci U S A. 1994 May 24;91(11):4950–4954. doi: 10.1073/pnas.91.11.4950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gasser S. M., Amati B. B., Cardenas M. E., Hofmann J. F. Studies on scaffold attachment sites and their relation to genome function. Int Rev Cytol. 1989;119:57–96. doi: 10.1016/s0074-7696(08)60649-x. [DOI] [PubMed] [Google Scholar]
  15. Giraldo R., Rhodes D. The yeast telomere-binding protein RAP1 binds to and promotes the formation of DNA quadruplexes in telomeric DNA. EMBO J. 1994 May 15;13(10):2411–2420. doi: 10.1002/j.1460-2075.1994.tb06526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Guschlbauer W., Chantot J. F., Thiele D. Four-stranded nucleic acid structures 25 years later: from guanosine gels to telomer DNA. J Biomol Struct Dyn. 1990 Dec;8(3):491–511. doi: 10.1080/07391102.1990.10507825. [DOI] [PubMed] [Google Scholar]
  17. Hager D. A., Burgess R. R. Elution of proteins from sodium dodecyl sulfate-polyacrylamide gels, removal of sodium dodecyl sulfate, and renaturation of enzymatic activity: results with sigma subunit of Escherichia coli RNA polymerase, wheat germ DNA topoisomerase, and other enzymes. Anal Biochem. 1980 Nov 15;109(1):76–86. doi: 10.1016/0003-2697(80)90013-5. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Hofmann J. F., Laroche T., Brand A. H., Gasser S. M. RAP-1 factor is necessary for DNA loop formation in vitro at the silent mating type locus HML. Cell. 1989 Jun 2;57(5):725–737. doi: 10.1016/0092-8674(89)90788-5. [DOI] [PubMed] [Google Scholar]
  21. Kilpatrick M. W., Torri A., Kang D. S., Engler J. A., Wells R. D. Unusual DNA structures in the adenovirus genome. J Biol Chem. 1986 Aug 25;261(24):11350–11354. [PubMed] [Google Scholar]
  22. Kim J., Ljungdahl P. O., Fink G. R. kem mutations affect nuclear fusion in Saccharomyces cerevisiae. Genetics. 1990 Dec;126(4):799–812. doi: 10.1093/genetics/126.4.799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kirk K. E., Harmon B. P., Reichardt I. K., Sedat J. W., Blackburn E. H. Block in anaphase chromosome separation caused by a telomerase template mutation. Science. 1997 Mar 7;275(5305):1478–1481. doi: 10.1126/science.275.5305.1478. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Lewis C. D., Clark S. P., Felsenfeld G., Gould H. An erythrocyte-specific protein that binds to the poly(dG) region of the chicken beta-globin gene promoter. Genes Dev. 1988 Jul;2(7):863–873. doi: 10.1101/gad.2.7.863. [DOI] [PubMed] [Google Scholar]
  26. Liu Z., Frantz J. D., Gilbert W., Tye B. K. Identification and characterization of a nuclease activity specific for G4 tetrastranded DNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3157–3161. doi: 10.1073/pnas.90.8.3157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Liu Z., Gilbert W. The yeast KEM1 gene encodes a nuclease specific for G4 tetraplex DNA: implication of in vivo functions for this novel DNA structure. Cell. 1994 Jul 1;77(7):1083–1092. doi: 10.1016/0092-8674(94)90447-2. [DOI] [PubMed] [Google Scholar]
  28. Ludérus M. E., van Steensel B., Chong L., Sibon O. C., Cremers F. F., de Lange T. Structure, subnuclear distribution, and nuclear matrix association of the mammalian telomeric complex. J Cell Biol. 1996 Nov;135(4):867–881. doi: 10.1083/jcb.135.4.867. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Makarov V. L., Hirose Y., Langmore J. P. Long G tails at both ends of human chromosomes suggest a C strand degradation mechanism for telomere shortening. Cell. 1997 Mar 7;88(5):657–666. doi: 10.1016/s0092-8674(00)81908-x. [DOI] [PubMed] [Google Scholar]
  30. Martindale D. W. Codon usage in Tetrahymena and other ciliates. J Protozool. 1989 Jan-Feb;36(1):29–34. doi: 10.1111/j.1550-7408.1989.tb02679.x. [DOI] [PubMed] [Google Scholar]
  31. Mazumder A., Neamati N., Ojwang J. O., Sunder S., Rando R. F., Pommier Y. Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures. Biochemistry. 1996 Oct 29;35(43):13762–13771. doi: 10.1021/bi960541u. [DOI] [PubMed] [Google Scholar]
  32. Mirzayan C., Copeland C. S., Snyder M. The NUF1 gene encodes an essential coiled-coil related protein that is a potential component of the yeast nucleoskeleton. J Cell Biol. 1992 Mar;116(6):1319–1332. doi: 10.1083/jcb.116.6.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moretti P., Freeman K., Coodly L., Shore D. Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1. Genes Dev. 1994 Oct 1;8(19):2257–2269. doi: 10.1101/gad.8.19.2257. [DOI] [PubMed] [Google Scholar]
  34. Pearson A. M., Rich A., Krieger M. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base-quartet-stabilized four-stranded helices. J Biol Chem. 1993 Feb 15;268(5):3546–3554. [PubMed] [Google Scholar]
  35. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pluta A. F., Kaine B. P., Spear B. B. The terminal organization of macronuclear DNA in Oxytricha fallax. Nucleic Acids Res. 1982 Dec 20;10(24):8145–8154. doi: 10.1093/nar/10.24.8145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Prescott D. M. The DNA of ciliated protozoa. Microbiol Rev. 1994 Jun;58(2):233–267. doi: 10.1128/mr.58.2.233-267.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sarig G., Weisman-Shomer P., Erlitzki R., Fry M. Purification and characterization of qTBP42, a new single-stranded and quadruplex telomeric DNA-binding protein from rat hepatocytes. J Biol Chem. 1997 Feb 14;272(7):4474–4482. doi: 10.1074/jbc.272.7.4474. [DOI] [PubMed] [Google Scholar]
  39. Schierer T., Henderson E. A protein from Tetrahymena thermophila that specifically binds parallel-stranded G4-DNA. Biochemistry. 1994 Mar 1;33(8):2240–2246. doi: 10.1021/bi00174a034. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Sen D., Gilbert W. Guanine quartet structures. Methods Enzymol. 1992;211:191–199. doi: 10.1016/0076-6879(92)11012-8. [DOI] [PubMed] [Google Scholar]
  42. Sundquist W. I., Heaphy S. Evidence for interstrand quadruplex formation in the dimerization of human immunodeficiency virus 1 genomic RNA. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3393–3397. doi: 10.1073/pnas.90.8.3393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. 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]
  44. Tishkoff D. X., Johnson A. W., Kolodner R. D. Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1. Mol Cell Biol. 1991 May;11(5):2593–2608. doi: 10.1128/mcb.11.5.2593. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Walsh K., Gualberto A. MyoD binds to the guanine tetrad nucleic acid structure. J Biol Chem. 1992 Jul 5;267(19):13714–13718. [PubMed] [Google Scholar]
  46. Wang K. Y., McCurdy S., Shea R. G., Swaminathan S., Bolton P. H. A DNA aptamer which binds to and inhibits thrombin exhibits a new structural motif for DNA. Biochemistry. 1993 Mar 2;32(8):1899–1904. doi: 10.1021/bi00059a003. [DOI] [PubMed] [Google Scholar]
  47. Weisman-Shomer P., Fry M. QUAD, a protein from hepatocyte chromatin that binds selectively to guanine-rich quadruplex DNA. J Biol Chem. 1993 Feb 15;268(5):3306–3312. [PubMed] [Google Scholar]
  48. Williamson J. R. G-quartet structures in telomeric DNA. Annu Rev Biophys Biomol Struct. 1994;23:703–730. doi: 10.1146/annurev.bb.23.060194.003415. [DOI] [PubMed] [Google Scholar]
  49. 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]
  50. Wright W. E., Tesmer V. M., Huffman K. E., Levene S. D., Shay J. W. Normal human chromosomes have long G-rich telomeric overhangs at one end. Genes Dev. 1997 Nov 1;11(21):2801–2809. doi: 10.1101/gad.11.21.2801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Zahler A. M., Williamson J. R., Cech T. R., Prescott D. M. Inhibition of telomerase by G-quartet DNA structures. Nature. 1991 Apr 25;350(6320):718–720. doi: 10.1038/350718a0. [DOI] [PubMed] [Google Scholar]

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