Enzymic removal of 5-methylcytosine from poly(dG-5-methyl-dC) by HeLa cell nuclear extracts is not by a DNA glycosylase.
A recent report in this journal [Vairapandi, M. and Duker, N.J. (1993) Nucleic Acids Res. 21, 5323-5327) presented evidence of an activity in HeLa cell nuclear extracts that released radiolabeled material from a poly(dG.dC) polymer that had been methylated and simultaneously labeled on cytosine residues by incubation with a CpG-specific DNA methylase and [methyl-3H]S-adenosylmethionine. Based on chromatographic evidence that the released products were thymine and 5-methylcytosine and on f1p4olabeling data suggesting a concomitant increase in abasic sites, the authors concluded that the releasing activity was a 5-methylcytosine-specific glycosylase and that the solubilized 5-methylcytosine was converted to thymine by a nuclear deaminase. We have confirmed that HeLa nuclear extracts promote release of ethanol-soluble radioactivity from a methyl-labeled poly(dG-5-methyl-dC)polymer, but the products released were neither 5-methylcytosine nor thymine. Furthermore, free 5-methylcytosine was not deaminated by incubation with the nuclear extract. The labeled compound released initially from the polymer appeared to be 5-methyl-deoxycytidine monophosphate, which was converted to 5-methyl-deoxycytidine, thymidine monophosphate, and/or thymidine by further incubation with the nuclear extract. The activity responsible for the release, therefore, was a nuclease. Release of 32P-labeled nucleotides from a 32P-labeled poly(dG-dC) polymer suggested, furthermore, that the activity was not specific for methylated DNA.
Steinberg
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RA
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
D044503:5-Methylcytosine; D002467:Cell Nucleus / Q000737:chemistry*; D003596:Cytosine / Q000031:analogs & derivatives* / Q000378:metabolism; D045647:DNA Glycosylases; D006367:HeLa Cells; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism
D011089:Polydeoxyribonucleotides; D011108:Polymers; C094101:poly(dG-5-methyl-dC); D044503:5-Methylcytosine; D003596:Cytosine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases