Demethylation of DNA by purified chick embryo 5-methylcytosine-DNA glycosylase requires both protein and RNA.
We have previously purified and characterized a 5-methylcytosine (5-MeC)-DNA glycosylase from 12 day old chick embryos [Jost,J.P. et al. (1995) J. Biol. Chem. 270, 9734-9739]. The activity of the purified enzyme is abolished upon treatment with proteinase K and ribonuclease A. RNA copurifies with 5-MeC-DNA glycosylase activity throughout all chromatographic steps and preparative gel electrophoresis. RNA with a length of approximately 300-500 nucleotides was isolated from the gel purified enzyme. Upon extensive treatment with proteinase K, the gel eluted and labeled RNA did not show any significant change in molecular mass. The purified RNA incubated alone or in the presence of Mg2+and deoxyribonucleotide phosphates had no 5-MeC-DNA glycosylase or demethylating activities. However, activity of 5-MeC-DNA glycosylase could be restored when the purified RNA was incubated with the inactive protein, free of RNA.
Frémont
M
M
Friedrich Miescher Institute, PO Box 2543, CH-4002 Basel, Switzerland.
Siegmann
M
M
Gaulis
S
S
Matthies
R
R
Hess
D
D
Jost
J P
JP
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000201:enzymology*; D002642:Chick Embryo; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification / Q000378:metabolism*; D012259:Ribonuclease, Pancreatic; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D012313:RNA; D004247:DNA; D012259:Ribonuclease, Pancreatic; C093335:5-methylcytosine-DNA glycosylase; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D019286:Endopeptidase K