5-Methylcytosine content of rat hepatoma DNA substituted with bromodeoxyuridine

Abstract

The aim of these experiments was to test whether incorporation of bromodeoxyuridine into DNA affects DNA methylation. Rat hepatoma (HTC) cells in culture were labeled for two generations with [14C]bromodeoxyuridine and [3H]thymidine to yield DNA which was 2.1, 20.6, 52.6, and 95.0% bromodeoxyuridine-substituted in the newly made strands. The DNA then was fractionated into highly repetitive, moderately repetitive, and single copy sequences. As determined by a comparison of 14C and 3H counts per min, the percentage of substitution with bromodeoxyuridine was found to be the same in each repetition class. The 5-methylcytosine content of each fraction was determined using high pressure liquid chromatography. It was found that bromodeoxyuridine, even at a level of substitution into newly mad DNA of 95%, has no effect on the 5-methylcytosine content of DNA. At all levels of bromodeoxyuridine substitution, highly repetitive DNA has slightly more 5-methylcytosine (3.0% of total cytosine) than does single copy DNA or moderately repetitive DNA (2.3%). The 5-methylcytosine content of whole HTC DNA is the same as that of rat liver DNA (2.4%).