Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Nov 11;17(21):8611–8629. doi: 10.1093/nar/17.21.8611

How different DNA sequences are recognized by a DNA-binding protein: effects of partial proteolysis.

P C Supakar 1, X Y Zhang 1, S Githens 1, R Khan 1, K C Ehrlich 1, M Ehrlich 1
PMCID: PMC335031  PMID: 2685746

Abstract

MDBP is a sequence-specific DNA-binding protein from mammals that recognizes a variety of DNA sequences, all of which show much homology to a partially palindromic 14 base-pair consensus sequence. MDBP subjected to limited proteolysis and then incubated with various specific oligonucleotide duplexes yielded two types of complexes. The relative concentrations of these complexes varied greatly depending on how closely the MDBP site matched the consensus sequence. No such DNA sequence-specific differences in the types of complexes formed were seen with intact MDBP. Partial proteolysis also changed the relative affinity of MDBP for several of its binding sites. The nature of the two types of complexes formed from fragmented MDBP and DNA was studied by DNA competition assays, protein titration, site-directed mutagenesis, and dimethyl sulfate and missing base interference assays. The results suggest that, for some specific DNA sequences, half-site interactions with one MDBP subunit predominate and for others, strong interaction of two subunits with both half-sites readily occur.

Full text

PDF
8627

Images in this article

8614Image
on p.8614
8616Image
on p.8616
8617Image
on p.8617
8621Image
on p.8621
8623Image
on p.8623

Selected References

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

  1. Boshart M., Weber F., Jahn G., Dorsch-Häsler K., Fleckenstein B., Schaffner W. A very strong enhancer is located upstream of an immediate early gene of human cytomegalovirus. Cell. 1985 Jun;41(2):521–530. doi: 10.1016/s0092-8674(85)80025-8. [DOI] [PubMed] [Google Scholar]
  2. Brunelle A., Schleif R. F. Missing contact probing of DNA-protein interactions. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6673–6676. doi: 10.1073/pnas.84.19.6673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chalepakis G., Beato M. Hydroxyl radical interference: a new method for the study of protein-DNA interactions. Nucleic Acids Res. 1989 Feb 25;17(4):1783–1783. doi: 10.1093/nar/17.4.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Costa R. H., Draper K. G., Banks L., Powell K. L., Cohen G., Eisenberg R., Wagner E. K. High-resolution characterization of herpes simplex virus type 1 transcripts encoding alkaline exonuclease and a 50,000-dalton protein tentatively identified as a capsid protein. J Virol. 1983 Dec;48(3):591–603. doi: 10.1128/jvi.48.3.591-603.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Costa R. H., Grayson D. R., Xanthopoulos K. G., Darnell J. E., Jr A liver-specific DNA-binding protein recognizes multiple nucleotide sites in regulatory regions of transthyretin, alpha 1-antitrypsin, albumin, and simian virus 40 genes. Proc Natl Acad Sci U S A. 1988 Jun;85(11):3840–3844. doi: 10.1073/pnas.85.11.3840. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Derynck R., Jarrett J. A., Chen E. Y., Eaton D. H., Bell J. R., Assoian R. K., Roberts A. B., Sporn M. B., Goeddel D. V. Human transforming growth factor-beta complementary DNA sequence and expression in normal and transformed cells. Nature. 1985 Aug 22;316(6030):701–705. doi: 10.1038/316701a0. [DOI] [PubMed] [Google Scholar]
  7. Draper K. G., Devi-Rao G., Costa R. H., Blair E. D., Thompson R. L., Wagner E. K. Characterization of the genes encoding herpes simplex virus type 1 and type 2 alkaline exonucleases and overlapping proteins. J Virol. 1986 Mar;57(3):1023–1036. doi: 10.1128/jvi.57.3.1023-1036.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Geisler N., Weber K. Isolation of amino-terminal fragment of lactose repressor necessary for DNA binding. Biochemistry. 1977 Mar 8;16(5):938–943. doi: 10.1021/bi00624a020. [DOI] [PubMed] [Google Scholar]
  9. Ghazal P., Lubon H., Hennighausen L. Multiple sequence-specific transcription factors modulate cytomegalovirus enhancer activity in vitro. Mol Cell Biol. 1988 Apr;8(4):1809–1811. doi: 10.1128/mcb.8.4.1809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Huang L. H., Wang R., Gama-Sosa M. A., Shenoy S., Ehrlich M. A protein from human placental nuclei binds preferentially to 5-methylcytosine-rich DNA. Nature. 1984 Mar 15;308(5956):293–295. doi: 10.1038/308293a0. [DOI] [PubMed] [Google Scholar]
  11. Hwung Y. P., Wang L. H., Tsai S. Y., Tsai M. J. Differential binding of the chicken ovalbumin upstream promoter (COUP) transcription factor to two different promoters. J Biol Chem. 1988 Sep 15;263(26):13470–13474. [PubMed] [Google Scholar]
  12. Khan R., Zhang X. Y., Supakar P. C., Ehrlich K. C., Ehrlich M. Human methylated DNA-binding protein. Determinants of a pBR322 recognition site. J Biol Chem. 1988 Oct 5;263(28):14374–14383. [PubMed] [Google Scholar]
  13. Mather E. L. DNA-binding factors of B lymphoid cells are susceptible to limited proteolytic cleavage during nuclear extract preparation. Mol Cell Biol. 1988 Apr;8(4):1812–1815. doi: 10.1128/mcb.8.4.1812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Pabo C. O., Sauer R. T., Sturtevant J. M., Ptashne M. The lambda repressor contains two domains. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1608–1612. doi: 10.1073/pnas.76.4.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Pfeifer K., Prezant T., Guarente L. Yeast HAP1 activator binds to two upstream activation sites of different sequence. Cell. 1987 Apr 10;49(1):19–27. doi: 10.1016/0092-8674(87)90751-3. [DOI] [PubMed] [Google Scholar]
  16. Quinn J. P., McGeoch D. J. DNA sequence of the region in the genome of herpes simplex virus type 1 containing the genes for DNA polymerase and the major DNA binding protein. Nucleic Acids Res. 1985 Nov 25;13(22):8143–8163. doi: 10.1093/nar/13.22.8143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Riggs A. D., Bourgeois S., Cohn M. The lac repressor-operator interaction. 3. Kinetic studies. J Mol Biol. 1970 Nov 14;53(3):401–417. doi: 10.1016/0022-2836(70)90074-4. [DOI] [PubMed] [Google Scholar]
  18. Sauer R. T., Pabo C. O., Meyer B. J., Ptashne M., Backman K. C. Regulatory functions of the lambda repressor reside in the amino-terminal domain. Nature. 1979 May 31;279(5712):396–400. doi: 10.1038/279396a0. [DOI] [PubMed] [Google Scholar]
  19. Schreiber E., Matthias P., Müller M. M., Schaffner W. Identification of a novel lymphoid specific octamer binding protein (OTF-2B) by proteolytic clipping bandshift assay (PCBA). EMBO J. 1988 Dec 20;7(13):4221–4229. doi: 10.1002/j.1460-2075.1988.tb03319.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Siebenlist U., Gilbert W. Contacts between Escherichia coli RNA polymerase and an early promoter of phage T7. Proc Natl Acad Sci U S A. 1980 Jan;77(1):122–126. doi: 10.1073/pnas.77.1.122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Singh H., LeBowitz J. H., Baldwin A. S., Jr, Sharp P. A. Molecular cloning of an enhancer binding protein: isolation by screening of an expression library with a recognition site DNA. Cell. 1988 Feb 12;52(3):415–423. doi: 10.1016/s0092-8674(88)80034-5. [DOI] [PubMed] [Google Scholar]
  22. Supakar P. C., Weist D., Zhang D. L., Inamdar N., Zhang X. Y., Khan R., Ehrlich K. C., Ehrlich M. Methylated DNA-binding protein is present in various mammalian cell types. Nucleic Acids Res. 1988 Aug 25;16(16):8029–8044. doi: 10.1093/nar/16.16.8029. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Tsai S. Y., Carlstedt-Duke J., Weigel N. L., Dahlman K., Gustafsson J. A., Tsai M. J., O'Malley B. W. Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell. 1988 Oct 21;55(2):361–369. doi: 10.1016/0092-8674(88)90059-1. [DOI] [PubMed] [Google Scholar]
  24. Tsai S. Y., Tsai M. J., O'Malley B. W. Cooperative binding of steroid hormone receptors contributes to transcriptional synergism at target enhancer elements. Cell. 1989 May 5;57(3):443–448. doi: 10.1016/0092-8674(89)90919-7. [DOI] [PubMed] [Google Scholar]
  25. Vinson C. R., LaMarco K. L., Johnson P. F., Landschulz W. H., McKnight S. L. In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage. Genes Dev. 1988 Jul;2(7):801–806. doi: 10.1101/gad.2.7.801. [DOI] [PubMed] [Google Scholar]
  26. Wang R. Y., Zhang X. Y., Ehrlich M. A human DNA-binding protein is methylation-specific and sequence-specific. Nucleic Acids Res. 1986 Feb 25;14(4):1599–1614. doi: 10.1093/nar/14.4.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wang R. Y., Zhang X. Y., Khan R., Zhou Y. W., Huang L. H., Ehrlich M. Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs. Nucleic Acids Res. 1986 Dec 22;14(24):9843–9860. doi: 10.1093/nar/14.24.9843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Zhang X. Y., Ehrlich K. C., Wang R. Y., Ehrlich M. Effect of site-specific DNA methylation and mutagenesis on recognition by methylated DNA-binding protein from human placenta. Nucleic Acids Res. 1986 Nov 11;14(21):8387–8397. doi: 10.1093/nar/14.21.8387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Zhang X. Y., Supakar P. C., Khan R., Ehrlich K. C., Ehrlich M. Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta. Nucleic Acids Res. 1989 Feb 25;17(4):1459–1474. doi: 10.1093/nar/17.4.1459. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES