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Nucleic Acids Research, 1995, Vol. 23, No. 9 1621-1624
© 1995

ENZYMOLOGY

Enzymic removal of 5-methylcytosine from poly(dG-5-methyl-dC) by HeLa cell nuclear extracts is not by a DNA glycosylase

Robert A. Steinberg

Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center PO Box 26901, Oklahoma City, OK 73190, USA

Received November 21, 1994. Revised March 18, 1995. Accepted March 18, 1995.

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 radlolabeled material from a poly(dG dC) polymer that had been methylated and simultaneously labeled on cytoslne residues by Incubation with a CpG-speclflc DNA methylase and [methyl-3H]S-adenosylmethionine. Based on chromato-graphic evidence that the released products were thymlne and 5-methylcytosine and on radiolabellng data suggesting a concomitant increase in abasic sites, the authors concluded that the releasing activity was a 5-methylcytosine-speclflc glycosylase and that the solubllized 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 thymlne. Furthermore, free 5-methylcytoslne was not deamlnated by Incubation with the nuclear extract. The labeled compound released initially from the polymer appeared to be 5-methyldeoxycytldine monophosphate, which was converted to 5-methyl-deoxycytidlne, thymldine monophosphate, and/or thymidlne by further incubation with the nuclear extract. The activity responsible for the release, therefore, was a nuclease. Release of 32P-labeled nucleo-tides from a 32P-labeled poly(dG-dC) polymer suggested, furthermore, that the activity was not specific for methylated DNA.


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