CHARACTERIZATION OF PROTEIN-AND DNA-INTERACTING DOMAINS IN HUMAN DNA (CYTOSINE-5)-METHYLTRANSFERASE

Abstract

Methylation of the carbon atom at the 5-position of cytosine is an important form of DNA modification in mammals. A unique feature of DNA methylation is its ability to serve as cellular memory, ensuring that somatic daughter cells retain parental phenotype. This is mediated by its role in gene regulation: DNA methylation is associated with imprinting, differentiation, embryonic development and genomic stability. Cytosine methylation was propelled into the cancer research upon discovery of abnormal methylation in the genomes of cancer cells. However, whether methylation is a causative factor in tumorigenesis remains unknown. Since DNA (cytosine-5) methyltransferase (MCMT) is the only known enzyme to effect the transfer of the methyl group from S-adenosyl-L- methionine lo the cytosine residue in mammalian DNA, understanding the intricacies of methylation would require a detailed knowledge of the relatively unexplored fictions of MCMT. The aims of this thesis focus on the functional characterization of individual domains in the human MCMT. First, the MCMT cDNA was isolated and expressed as a recombinant protein which was used to develop specific antibodies for Western analysis. Interestingly, Western analyses of various human cell extracts showed that MCMT was truncated during apoptosis and specifically in HL-60 cells under normal growth conditions. These results offer an idea of potentially informative domains in the relatively large 190kD protein to be studied.