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. 1993 May 25;21(10):2339–2342. doi: 10.1093/nar/21.10.2339

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

S Kochanek 1, D Renz 1, W Doerfler 1
PMCID: PMC309529  PMID: 8506131

Abstract

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.

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Selected References

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  1. Degen S. J., Rajput B., Reich E. The human tissue plasminogen activator gene. J Biol Chem. 1986 May 25;261(15):6972–6985. [PubMed] [Google Scholar]
  2. Dignam J. D., Lebovitz R. M., Roeder R. G. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 1983 Mar 11;11(5):1475–1489. doi: 10.1093/nar/11.5.1475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Drew H. R. Structural specificities of five commonly used DNA nucleases. J Mol Biol. 1984 Jul 15;176(4):535–557. doi: 10.1016/0022-2836(84)90176-1. [DOI] [PubMed] [Google Scholar]
  4. Drew H. R., Travers A. A. DNA structural variations in the E. coli tyrT promoter. Cell. 1984 Jun;37(2):491–502. doi: 10.1016/0092-8674(84)90379-9. [DOI] [PubMed] [Google Scholar]
  5. Geiduschek E. P., Tocchini-Valentini G. P. Transcription by RNA polymerase III. Annu Rev Biochem. 1988;57:873–914. doi: 10.1146/annurev.bi.57.070188.004301. [DOI] [PubMed] [Google Scholar]
  6. Jüttermann R., Hosokawa K., Kochanek S., Doerfler W. Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation. J Virol. 1991 Apr;65(4):1735–1742. doi: 10.1128/jvi.65.4.1735-1742.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Klenow H., Overgaard-Hansen K., Patkar S. A. Proteolytic cleavage fo native DNA polymerase into two different catalytic fragments. Influence of assay condtions on the change of exonuclease activity and polymerase activity accompanying cleavage. Eur J Biochem. 1971 Oct 14;22(3):371–381. doi: 10.1111/j.1432-1033.1971.tb01554.x. [DOI] [PubMed] [Google Scholar]
  8. Kochanek S., Renz D., Doerfler W. DNA methylation in the Alu sequences of diploid and haploid primary human cells. EMBO J. 1993 Mar;12(3):1141–1151. doi: 10.1002/j.1460-2075.1993.tb05755.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kurachi K., Davie E. W., Strydom D. J., Riordan J. F., Vallee B. L. Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor. Biochemistry. 1985 Sep 24;24(20):5494–5499. doi: 10.1021/bi00341a032. [DOI] [PubMed] [Google Scholar]
  10. LEHMAN I. R., BESSMAN M. J., SIMMS E. S., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem. 1958 Jul;233(1):163–170. [PubMed] [Google Scholar]
  11. Lahm A., Suck D. DNase I-induced DNA conformation. 2 A structure of a DNase I-octamer complex. J Mol Biol. 1991 Dec 5;222(3):645–667. doi: 10.1016/0022-2836(91)90502-w. [DOI] [PubMed] [Google Scholar]
  12. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ohmori H., Tomizawa J. I., Maxam A. M. Detection of 5-methylcytosine in DNA sequences. Nucleic Acids Res. 1978 May;5(5):1479–1485. doi: 10.1093/nar/5.5.1479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Perez-Stable C., Ayres T. M., Shen C. K. Distinctive sequence organization and functional programming of an Alu repeat promoter. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5291–5295. doi: 10.1073/pnas.81.17.5291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Perez-Stable C., Shen C. K. Competitive and cooperative functioning of the anterior and posterior promoter elements of an Alu family repeat. Mol Cell Biol. 1986 Jun;6(6):2041–2052. doi: 10.1128/mcb.6.6.2041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Renbaum P., Abrahamove D., Fainsod A., Wilson G. G., Rottem S., Razin A. Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp. strain MQ1(M.SssI). Nucleic Acids Res. 1990 Mar 11;18(5):1145–1152. doi: 10.1093/nar/18.5.1145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Richardson C. C. Phosphorylation of nucleic acid by an enzyme from T4 bacteriophage-infected Escherichia coli. Proc Natl Acad Sci U S A. 1965 Jul;54(1):158–165. doi: 10.1073/pnas.54.1.158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Suck D., Oefner C. Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA. Nature. 1986 Jun 5;321(6070):620–625. doi: 10.1038/321620a0. [DOI] [PubMed] [Google Scholar]
  20. Toth M., Müller U., Doerfler W. Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter. J Mol Biol. 1990 Aug 5;214(3):673–683. doi: 10.1016/0022-2836(90)90285-T. [DOI] [PubMed] [Google Scholar]
  21. Yoshinaga S. K., L'Etoile N. D., Berk A. J. Purification and characterization of transcription factor IIIC2. J Biol Chem. 1989 Jun 25;264(18):10726–10731. [PubMed] [Google Scholar]

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