Various roles have been proposed for the methylation and demethylation of cytosines in eucaryotic DNA. These possible roles include: (1) a transcriptional control mechanism, (2) a modification system that protects specific pieces of DNA from destruction, (3) maintenance or loss of the latent state of viral genomes in both transformed and nontransformed cells, and (4) control of initiation of DNA synthesis. In the specific case of the lymphotropic herpesviruses, many of which generate tumors of the lymphatic system in a variety of animals, evidence has accumulated showing DNA methylation/demethylation to be a critical component of the shifts between latent and productive phases of the viral life cycle. In the work proposed here, transformed lymphocytes infected with the herpesvirus Epstein-Barr will be the experimental system employed to determine the mechanism of DNA methylation and demethylation. Both viral and cellular DNA isolated from these cell lines show highly variable degrees of methylation at many CG sites. We propose here to identify, isolate and purify methylases (DNA methyl transferases) from these lymphocytes and test for possible sequence preferences, variable affinities for viral and cellular DNA, and whether such activities are virally induced or repressed. We further propose to develop three separate assay systems to identify the DNA demethylating mechanism(s) operating within these cells.
