Distribution of 5-Methyldeoxycytidine in Products of Staphylococcal Nuclease Digestion of Nuclei and Purified DNA

We have compared the distribution of 5-methyldeoxycytidine (m⁵dC) between staphylococcal nuclease (SN) sensitive and resistant regions of human diploid fibroblast chromatin to the corresponding distribution in purified DNA. After SN digestion of fibroblast nuclei or purified DNA, nuclease-resistant products were separated from sensitive products by perchloric acid or ethanol precipitation; the radioactively labeled nucleosides were then fractionated by high-performance liquid chromatography and quantitated. Our results indicate that m⁵dC is preferentially associated with SN-resistant regions of both chromatin and purified DNA. The magnitudes of these preferences in fibroblast chromatin and DNA are similar; we find that the enrichment of m⁵dC content in SN-resistant fractions of nuclei and DNA relative to the corresponding sensitive fractions is approximately 2-3-fold. Therefore, highly methylated regions of DNA have an intrinsic resistance to digestion by SN that is of sufficient magnitude to explain the high degree of nuclease resistance of chromatin containing highly methylated DNA.