Skip to main content
SpringerLink
Log in

Kinetic mechanisms and interaction of rat liver DNA methyltransferase with defined DNA substrates

  • Original Article
  • Published:
Molecular and Cellular Biochemistry Aims and scope Submit manuscript

Abstract

DNA substrate analogs were constructed from poly(dC-dG), M13, and XP12 DNA which do not contain a mixture of types of methylation sites. These were used to distinguish different kinetic mechanisms for maintenance and de novo methylation using a highly purified rat liver DNA (cytosine-5)-methyltransferase (DMase−) preparation. De novo methylation on single (ss) and double-stranded (ds) DNA was found to obey Michaelis-Menten kinetics while methylation of hemimethylated sites showed differences depending on size of the hemimethylated region. On long stretches analogous to maintenance methylation of newly replicated DNA, saturation could not be achieved and the kinetics showed non-ideal positive cooperative kinetics, while short stretches showed non-Michaelis-Menten kinetics and rapid saturation. Two types of DMase-DNA complexes could be distinguished by means of affinity chromatography on DNA-agarose matrices and in preincubation assays. The later complex, which is engaged in methyl group turnover, exhibited enhanced stability. The competitiveness of variously configured DNAs was found to parallel the stability of complex formation, e.g., ss, hemi- and ds DNA, respectively. In studies utilizing 5-bromodeoxyuridine, the thymine analog left the basic reaction mechanisms unchanged but increased the km and S0.5 while reducing the velocity of these reactions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

AzaC:

5-azacytidine

BrdU:

5-bromodeoxyuridine

DMase:

DNA (cytosine-5) methyltransferase (EC 2.1.1.37)

hemi:

hemimethylated

kd:

kilodalton

M-M:

Michaelis-Menten

SAM:

S-adenosyl-L-methionine

SDS:

sodium dodecylsulfate

S0.5 :

substrate concentration at half maximal velocity of reaction (Vmax)

ss:

single-stranded

ds:

double-stranded

References

  1. Adams RLP, Burdon RH: CRC Crit Rev Biochem 13:349–384, 1982

    Google Scholar 

  2. Lapeyre J-N, Ruchirawat M, Becker FE In: Bekhor I (ed), Progress in Non-Histone Protein Research, Vol I, pp 167–190 CRC Press Inc, West Palm Beach, Florida, 1985

    Google Scholar 

  3. Adams RLP: Biochim Biophys Acta 254:205–212, 1971

    Google Scholar 

  4. Bird AP: J Mol Biol 118:49–60, 1978

    Google Scholar 

  5. Jahner D, Jaenisch R: Nature 315:594–597, 1985

    Google Scholar 

  6. Razin A, Webb C, Szyf M, Yisraeli J, Rosenthal A, Naveh-Many T, Sciaky-Gallili N, Cedar H: Proc Natl Acad Sci USA 81:2275–2279, 1984

    Google Scholar 

  7. Groudine M, Conklin KF: Science 228:1061–1068, 1985

    Google Scholar 

  8. Hilliard JK, Sneider TW: Nucl Acids Res 2:809–819, 1975

    Google Scholar 

  9. Mohandas T, Sparkes RS, Shapiro LJ: Science 211:393–396, 1981

    Google Scholar 

  10. Wigler M, Levy D, Perucho M: Cell 24:33–40, 1981

    Google Scholar 

  11. Doerfler W: Ann Rev Biochem 52:92–124, 1983

    Google Scholar 

  12. Felsenfeld G, McGhee J: Nature 296:602–603, 1982

    Google Scholar 

  13. Razin A, Friedman S: Prog Nucleic Acids Res Mol Biol 25:33–52, 1981

    Google Scholar 

  14. Busslinger M, Hurst G, Flavell R: Cell 34:197–206, 1983

    Google Scholar 

  15. Keshet I, Yisraeli J, Cedar H: Proc Natl Acad Sci USA 83:2560–2564, 1985

    Google Scholar 

  16. Bestor TH, Ingram VM: Proc Natl Acad Sci USA 80:5559–5563, 1983

    Google Scholar 

  17. Pfeifer GP, Grunwald S, Palitti F, Kaul S, Boehm TLJ, Hirth HP, Drahovsky D: J Biol Chem 260:13787–13793, 1985

    Google Scholar 

  18. Christman JK, Price P, Pedrinan L, Acs G: Eur J Biochem 83:53, 1979

    Google Scholar 

  19. Sneider TW, Teague WM, Rogachevsky LM: Nucl Acids Res 2:1685–1700, 1975

    Google Scholar 

  20. Jones PA, Taylor SM: Nucleic Acids Res. 9:2933–2947, 1981

    Google Scholar 

  21. Taylor SM, Jones PA: J Mol Biol 162:679–692, 1982

    Google Scholar 

  22. Christman SK, Schneiderman N, Acs G: J Biol Chem. 260:4059–4064, 1985

    Google Scholar 

  23. Lapeyre J-N, Bekhor I: J Mol Biol 104:25–58, 1976

    Google Scholar 

  24. Gruenbaum V, Cedar M, Razin A: Nature 295:620–622, 1982

    Google Scholar 

  25. Wang RYH, Huang LH, Ehrlich M: Nucl. Acids Res 12:3473–3490, 1984

    Google Scholar 

  26. Becker FF, Holton R, Ruchirawat M Lapeyre J-N: Proc Natl Acad Science USA 82:6055–6059, 1985

    Google Scholar 

  27. Ruchirawatt M, Becker FF, Lapeyre J-N: Carcinogenesis 6:877–882, 1985

    Google Scholar 

  28. Ruchirawat M, Becker FF, Lapeyre J-N: Biochemistry 23:5426–5432, 1984

    Google Scholar 

  29. Messing J, Viera J: Gene 19:269–276, 1982

    Google Scholar 

  30. Lapeyre J-N, Becker FF: Biochem Biophys Res Comm 87:698–705, 1979

    Google Scholar 

  31. Arndt-Jovin DJ, Jovin TM, Bahr W, Brischanf A-M, Marguardt M: Eur J Biochem 54:411–418, 1975

    Google Scholar 

  32. Singer J, Stellwagen RH, Robert-Ems J, Riggs AD: J Biol Chem 252:5509–5513, 1977

    Google Scholar 

  33. Ricard J, Noat J: Eur J Biochem 152:557–564, 1985

    Google Scholar 

  34. Drahovsky D, Morris RN: J Mol Biol 61:343–356, 1971

    Google Scholar 

  35. Ruchirawat M, Becker FF, Lapeyre J-N: Nucl Acids Res 12:3357–3372, 1984

    Google Scholar 

  36. Adams RLP, McKay EL, Graig LM, Burdon RH: Biochim Biophys Acta 561:345–357, 1979

    Google Scholar 

  37. Simon D, Grunert F, Acken UV, Doting HP, Kroger H: Nucl Acids Res 6:2153–2167, 1978

    Google Scholar 

  38. Roy PH, Weissbach A: Nucl Acids Res 2:1669–1684, 1975

    Google Scholar 

  39. Pfeiffer GP, Grunwald S, Boehm TLJ, Drahovsky D: Biochim Biophys Acta 740:323–330, 1983

    Google Scholar 

  40. Carotti D, Palitti F, Mastrantonio S, Rispoli M, Strom R, Amato A, Campagnari F, Whitehead EP: Biochem Biophys Acta 866:135–143, 1986

    Google Scholar 

  41. Sano H, Noguchi H, Sager R: Eur J Biochem 135:181–185, 1983

    Google Scholar 

  42. Adams RP, Burdon RH, McKinnon K, Rinaldi A: FEBS Lett 163:194–198, 1983

    Google Scholar 

  43. Davis T, Kirk D, Rinaldi A, Burdon RH, Adams RLP: Biochem Biophys Res Comm 126:678–684, 1985

    Google Scholar 

  44. Burdon RH, Qureshi M, Adams RLP: Biochim Biophys Acta 825:70–79, 1985

    Google Scholar 

  45. Bolden A, Ward C, Siedlecki JA, Weissbach A: J Biol Chem 259:12437–12443, 1984

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology, Section of Experimental Pathology, The University of Texas M.D. Anderson Hospital and Tumor Institute at Houston, 6723 Bertner Avenue, 77030, Houston, Texas, USA

    Mathuros Ruchirawat, Jamileh Noshari & Jean-Numa Lapeyre

Authors
  1. Mathuros Ruchirawat
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jamileh Noshari
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Numa Lapeyre
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ruchirawat, M., Noshari, J. & Lapeyre, JN. Kinetic mechanisms and interaction of rat liver DNA methyltransferase with defined DNA substrates. Mol Cell Biochem 76, 45–54 (1987). https://doi.org/10.1007/BF00219397

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00219397

Key words

Search

Navigation