INFIDELITY OF CYTOSINE METHYLATION AND HUMAN CANCER

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Emerging data strongly suggests that the infidelity of DNA cytosine methyltransferase may result in the perturbation of cytosine methylation patterns and contribute to the development of cancer in man. Altered cytosine methytransferase activity may result in activation of oncogenes, silencing of tumor suppressor genes, or mutation through induced cytosine deamination. Several methods have been developed which allow determination of the methylation status of specific cytosine residues in DNA derived from cell lines and tumor specimens. Substantially less attention, however, has been focused on the mechanisms by which methylation patterns may become altered or perturbed. We have developed a novel in vitro assay, which allows us to determine quantitatively the DNA substrate preferences for DNA cytosine methyltransferases. This method, which we call mass tagging, is based upon labelling target cytosine residues in synthetic oligonucleotides with stable isotopes. Methylase-mediated base modification is examined by gas chromatography/mass spectrometry (UC/MS). With this method, we can examine the effects of DNA structural perturbations, such as mispairs and damaged bases, on methylase rate and base selectivity. We can also examine the effects of oligonucleotides bearing mechanism-based inhibitors such as 5-fluorocytosine and 5-azacytosine, and the tendency of the maintenance methylase to methylate unmethylated sites adjacent to hemimethylated sites. We propose to study purified mammalian maintenance methylases, a newly identified class of "de novo" methylases, as well as extracts from cells with demonstrated perturbations in methylation patterns. Completion of the proposed studies will substantially increase our understanding of the mechanisms by which the fidelity of cytosine methylation may be reduced and provide tools for testing agents which may increase the fidelity of methylation in vivo or act as selective methylase inhibitors.