Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1984 Apr 11;12(7):3357–3372. doi: 10.1093/nar/12.7.3357

Interaction of DNA methyltransferase with aminofluorene and N-acetylaminofluorene modified poly(dC-dG).

M Ruchirawat, F F Becker, J N Lapeyre
PMCID: PMC318751  PMID: 6718252

Abstract

Poly(dC-dG) was reacted in vitro to yield templates containing similar amounts of aminofluorene (AF) or acetylaminofluorene (AAF) adducts. These modified poly(dC-dG) templates were tested in an in vitro DNA methylation system utilizing 1500-fold purified rat liver methyltransferase (DMase) to compare and quantitate the effects of these adducts on the kinetics of methylation and the interaction of DMase with such templates. Enzymatic methylation is severely impaired by arylamine adducts, with bound AF inhibiting more than AAF (relative Vmax 0.24 for AAF-poly(dC-dG) and 0.066 for AF-poly(dC-dG). The apparent km for the reaction is not appreciably altered by AAF modification: 10 microM for dCdG dinucleotide units, but it is threefold lower (3 microM) for AF-poly(dC-dG). In competition experiments it was demonstrated that a translocational block is imposed by the adducts. From differential salt inhibition assays and preincubation assays, no change in the ionic binding to the altered templates could be detected, which suggests that the enzyme interacts very strongly through hydrophobic interactions with the fluorene ring. Evidence that the fluorene ring is exposed is supported by circular dichroism spectra of the templates under the conditions of the assay, which indicated that the AF adducts do not appreciably change the normal B conformation of the template, while the template with 9.5% modification by AAF adducts adopted a Z form. These results suggest that the inhibitory effects of AAF and, in particular, AF upon DMase-catalyzed methylation reactions are not dependent upon helix conformation. Instead, they appear to depend upon DMase recognition of an altered dG base configuration, which is responsible for altered binding and methylation kinetics.

Full text

PDF
3357

Selected References

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

  1. Arnott S., Chandrasekaran R., Birdsall D. L., Leslie A. G., Ratliff R. L. Left-handed DNA helices. Nature. 1980 Feb 21;283(5749):743–745. doi: 10.1038/283743a0. [DOI] [PubMed] [Google Scholar]
  2. Behe M., Felsenfeld G. Effects of methylation on a synthetic polynucleotide: the B--Z transition in poly(dG-m5dC).poly(dG-m5dC). Proc Natl Acad Sci U S A. 1981 Mar;78(3):1619–1623. doi: 10.1073/pnas.78.3.1619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bird A. P. DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res. 1980 Apr 11;8(7):1499–1504. doi: 10.1093/nar/8.7.1499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boehm T. L., Grunberger D., Drahovsky D. Aberrant de novo methylation of DNA after treatment of murine cells with N-acetoxy-N-2-acetylaminofluorene. Cancer Res. 1983 Dec;43(12 Pt 1):6066–6071. [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Browne M. J., Burdon R. H. The sequence specificity of vertebrate DNA methylation. Nucleic Acids Res. 1977 Apr;4(4):1025–1037. doi: 10.1093/nar/4.4.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. DeBaun J. R., Smith J. Y., Miller E. C., Miller J. A. Reactivity in vivo of the carcinogen N-hydroxy-2-acetylaminofluorene: increase by sulfate ion. Science. 1970 Jan 9;167(3915):184–186. doi: 10.1126/science.167.3915.184. [DOI] [PubMed] [Google Scholar]
  8. Drahovský D., Morris N. R. Mechanism of action of rat liver DNA methylase. II. Interaction with single-stranded methyl-acceptor DNA. J Mol Biol. 1971 Oct 28;61(2):343–356. doi: 10.1016/0022-2836(71)90384-6. [DOI] [PubMed] [Google Scholar]
  9. Ehrlich M., Wang R. Y. 5-Methylcytosine in eukaryotic DNA. Science. 1981 Jun 19;212(4501):1350–1357. doi: 10.1126/science.6262918. [DOI] [PubMed] [Google Scholar]
  10. Feinberg A. P., Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983 Jan 6;301(5895):89–92. doi: 10.1038/301089a0. [DOI] [PubMed] [Google Scholar]
  11. Holliday R. A new theory of carcinogenesis. Br J Cancer. 1979 Oct;40(4):513–522. doi: 10.1038/bjc.1979.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Irving C. C. Enzymatic deacetylation of N-hydroxy-2-acetylaminofluorene by liver microsomes. Cancer Res. 1966 Jul;26(7):1390–1396. [PubMed] [Google Scholar]
  13. King C. M. Mechanism of reaction, tissue distribution, and inhibition of arylhydroxamic acid acyltransferase. Cancer Res. 1974 Jun;34(6):1503–1515. [PubMed] [Google Scholar]
  14. Kriek E., Miller J. A., Juhl U., Miller E. C. 8-(N-2-fluorenylacetamido)guanosine, an arylamidation reaction product of guanosine and the carcinogen N-acetoxy-N-2-fluorenylacetamide in neutral solution. Biochemistry. 1967 Jan;6(1):177–182. doi: 10.1021/bi00853a029. [DOI] [PubMed] [Google Scholar]
  15. Kriek E. On the mechanism of action of carcinogenic aromatic amines. I. Binding of 2-acetylaminofluorene and N-hydroxy-2-acetylaminofluorene to rat-liver nucleic acids in vivo. Chem Biol Interact. 1969 Oct;1(1):3–17. doi: 10.1016/0009-2797(69)90015-5. [DOI] [PubMed] [Google Scholar]
  16. Kriek E. Persistent binding of a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo. Cancer Res. 1972 Oct;32(10):2042–2048. [PubMed] [Google Scholar]
  17. Lapeyre J. N., Becker F. F. 5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result. Biochem Biophys Res Commun. 1979 Apr 13;87(3):698–705. doi: 10.1016/0006-291x(79)92015-1. [DOI] [PubMed] [Google Scholar]
  18. Lapeyre J. N., Walker M. S., Becker F. F. DNA methylation and methylase levels in normal and malignant mouse hepatic tissues. Carcinogenesis. 1981;2(9):873–878. doi: 10.1093/carcin/2.9.873. [DOI] [PubMed] [Google Scholar]
  19. Lefèvre J. F., Fuchs R. P., Daune M. P. Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides. Biochemistry. 1978 Jun 27;17(13):2561–2567. doi: 10.1021/bi00606a016. [DOI] [PubMed] [Google Scholar]
  20. Levine A. F., Fink L. M., Weinstein I. B., Grunberger D. Effect of N-2-acetylaminofluorene modification on the conformation of nucleic acids. Cancer Res. 1974 Feb;34(2):319–327. [PubMed] [Google Scholar]
  21. Miller E. C. Some current perspectives on chemical carcinogenesis in humans and experimental animals: Presidential Address. Cancer Res. 1978 Jun;38(6):1479–1496. [PubMed] [Google Scholar]
  22. Moore P. D., Rabkin S. D., Osborn A. L., King C. M., Strauss B. S. Effect of acetylated and deacetylated 2-aminofluorene adducts on in vitro DNA synthesis. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7166–7170. doi: 10.1073/pnas.79.23.7166. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Möller A., Nordheim A., Nichols S. R., Rich A. 7-Methylguanine in poly(dG-dC).poly(dG-dC) facilitates z-DNA formation. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4777–4781. doi: 10.1073/pnas.78.8.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Pfohl-Leszkowicz A., Boiteux S., Laval J., Keith G., Dirheimer G. Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms. Biochem Biophys Res Commun. 1983 Oct 31;116(2):682–688. doi: 10.1016/0006-291x(83)90579-x. [DOI] [PubMed] [Google Scholar]
  25. Pfohl-Leszkowicz A., Salas C., Fuchs R. P., Dirheimer G. Mechanism of inhibition of enzymatic deoxyribonucleic acid methylation by 2-(acetylamino)fluorene bound to deoxyribonucleic acid. Biochemistry. 1981 May 26;20(11):3020–3024. doi: 10.1021/bi00514a005. [DOI] [PubMed] [Google Scholar]
  26. Pohl F. M., Jovin T. M. Salt-induced co-operative conformational change of a synthetic DNA: equilibrium and kinetic studies with poly (dG-dC). J Mol Biol. 1972 Jun 28;67(3):375–396. doi: 10.1016/0022-2836(72)90457-3. [DOI] [PubMed] [Google Scholar]
  27. Razin A., Friedman J. DNA methylation and its possible biological roles. Prog Nucleic Acid Res Mol Biol. 1981;25:33–52. doi: 10.1016/s0079-6603(08)60482-1. [DOI] [PubMed] [Google Scholar]
  28. Reilly J. G., Thomas C. A., Jr, Sen A. DNA methylation in mouse cells in culture as measured by restriction enzymes. Biochim Biophys Acta. 1982 Apr 26;697(1):53–59. doi: 10.1016/0167-4781(82)90044-6. [DOI] [PubMed] [Google Scholar]
  29. Sage E., Leng M. Conformation of poly(dG-dC) . poly(dG-dC) modified by the carcinogens N-acetoxy-N-acetyl-2-aminofluorene and N-hydroxy-N-2-aminofluorene. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4597–4601. doi: 10.1073/pnas.77.8.4597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sage E., Leng M. Conformational changes of poly(dG-dC) . poly(dG-dC) modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. Nucleic Acids Res. 1981 Mar 11;9(5):1241–1250. doi: 10.1093/nar/9.5.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sakai S., Reinhold C. E., Wirth P. J., Thorgeirsson S. S. Mechanism of in vitro mutagenic activation and covalent binding of N-hydroxy-2-acetylaminofluorene in isolated liver cell nuclei from rat and mouse. Cancer Res. 1978 Jul;38(7):2058–2067. [PubMed] [Google Scholar]
  32. Salas C. E., Pfohl-Leszkowicz A., Lang M. C., Dirheimer G. Effect of modification by N-acetoxy-N-2-acetylaminofluorene on the level of DNA methylation. Nature. 1979 Mar 1;278(5699):71–72. doi: 10.1038/278071a0. [DOI] [PubMed] [Google Scholar]
  33. Santella R. M., Grunberger D., Weinstein I. B., Rich A. Induction of the Z conformation in poly(dG-dC).poly(dG-dC) by binding of N-2-acetylaminofluorene to guanine residues. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1451–1455. doi: 10.1073/pnas.78.3.1451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Simon D., Grunert F., von Acken U., Döring H. P., Kröger H. DNA-methylase from regenerating rat liver: purification and characterisation. Nucleic Acids Res. 1978 Jun;5(6):2153–2167. doi: 10.1093/nar/5.6.2153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Singer B. All oxygens in nucleic acids react with carcinogenic ethylating agents. Nature. 1976 Nov 25;264(5584):333–339. doi: 10.1038/264333a0. [DOI] [PubMed] [Google Scholar]
  36. Spodheim-Maurizot M., Saint-Ruf G., Leng M. Conformational changes induced in DNA by in vitro reaction with N-hydroxy-N-2-aminofluorene. Nucleic Acids Res. 1979 Apr;6(4):1683–1694. doi: 10.1093/nar/6.4.1683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Stout D. L., Hemminki K., Becker F. F. Covalent binding of 2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-acetylaminofluorene to rat liver nuclear DNA and protein in vivo and in vitro. Cancer Res. 1980 Oct;40(10):3579–3584. [PubMed] [Google Scholar]
  38. Straub K. M., Meehan T., Burlingame A. L., Calvin M. Identification of the major adducts formed by reaction of benzo(a)pyrene diol epoxide with DNA in vitro. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5285–5289. doi: 10.1073/pnas.74.12.5285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Westra J. G., Kriek E., Hittenhausen H. Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo. Chem Biol Interact. 1976 Oct 2;15(2):149–164. doi: 10.1016/0009-2797(76)90160-5. [DOI] [PubMed] [Google Scholar]
  40. Westra J. G., Visser A. Quantitative analysis of N-(guanin-8-yl)-N-acetyl-2-aminofluorene and N-(guanin-8-yl)-2-aminofluorene in modified DNA by hydrolysis in trifluoroacetic acid and high pressure liquid chromatography. Cancer Lett. 1979 Dec;8(2):155–162. doi: 10.1016/0304-3835(79)90009-0. [DOI] [PubMed] [Google Scholar]
  41. van de Sande J. H., Jovin T. M. Z* DNA, the left-handed helical form of poly[d(G-C)] in MgCl2-ethanol, is biologically active. EMBO J. 1982;1(1):115–120. doi: 10.1002/j.1460-2075.1982.tb01133.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van der Ploeg L. H., Flavell R. A. DNA methylation in the human gamma delta beta-globin locus in erythroid and nonerythroid tissues. Cell. 1980 Apr;19(4):947–958. doi: 10.1016/0092-8674(80)90086-0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES