ENZYMATIC 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) N ucleic 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 labe led on cytosine residues by incubation with a CpG-specific DNA methyla se and [methyl-H-3]S-adenosylmethionine. Based on chromatographic evid ence that the released products were thymine and 5-methylcytosine and on radiolabeling data suggesting a concomitant increase in abasic site s, the authors concluded that the releasing activity was a 5-methylcyt osine-specific glycosylase and that the solubilized 5-methylcytosine w as converted to thymine by a nuclear deaminase, We have confirmed that HeLa nuclear extracts promote release of ethanol-soluble radioactivit y 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 ext ract, The labeled compound released initially from the polymer appeare d to be 5-methyl-deoxycytidine, thymidine monophosphate, and/or thymid ine by further incubation with the nuclear extract, The activity respo nsible for the release, therefore, was a nuclease, Release of P-32-lab eled nucleotides from a P-32-labeled poly(dG . dC) polymer suggested, furthermore, that the activity was not specific for methylated DNA.