Vol. 57, No. 2, 1974 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS CORRELATION BETWEEN SYNTHESIS AND METHYLATION OF DNA IN HELA CELLS+ Domenico Geraci, Tamilla Eremenko, Roberta Cocchiara, Aldo Granieri, Eduardo Scarano and Pietro Volpe International Institute of Genetics and Biophysics, Naples, and Chair of Molecular Biology, University of Naples, Italy Received January 28, 1974 SUMMARY - For the whole cell cycle the methylation of DNA was studied in synchronized HeLa cells and in nuclei isolated from them. In the intact cells the methylation of DNA cytosine runs parallel to DNA synthesis. The pattern of DNA cytosine methylation by the isolated nuclei is almost identical to that obtained with the whole cells. Since the isolated nuclei do not synthesize DNA, it is shown that DNA methylation continues for at least 30 min after DNA syn- thesis is over. No DNA minor thymine is found in the isolated nuclei. In the cells of higher organisms the only minor methyl base found in DNA is 5-methylcytosine (l-3). In eukaryotes as in bacteria the methyl group of DNA 5-methylcytosine originates from the methyl group of SAM'. The mechanism of DNA methylation in vivo is, however, very poorly understood. Kappler (3) has recently suggested that in animal cell DNA methylation follows DNA synthesis by about 2 min. Other authors (5-8) have suggested that methyla- tion may continue for some hours after synthesis of DNA is completed. Isolated nuclei, which in the absence of deoxynucleoside triphosphates do not synthesize DNA (9), are able to methylate their own DNA (10-12). They ap- pear to be, thus, a useful tool for studying the mechanism of DNA methylation. In this paper experiments are reported on the correlation between DNA syn- thesis and DNA methylation in synchronized HeLa cells and in nuclei isolated from them at hourly intervals during the cell cycle. + These findings were presented at the 9th International Congress of Biochem- istry, July 1973, Stockholm, Sweden. Abstract book, 3mh4. 7 1 Abbreviations: Z4 . . 3H-sAM, PH-met~~~-ME~, L C-methyl-! L-methionine ; SAM, S-adenosyl-L-methionine; S adenosyl-L-methionine; SDS, Sodium dodecyl sulfate. Address: International Institute of Genetics and Biophysics, Via Marconi 10, 80125 Naples, Italy. 353