Radiation induced damage to 5-methylcytosine in DNA and its enzymatic repair

5-Methylcytosine is a minor base present in most eukaryotic DNA. It is a normal post-synthetic modification of cytosine and occurs primarily in CpG doublets. It is believed that methylation of CpG dinucleotides regulates gene expression. Recent findings also show that 5-methylcytosine contributes to the carcinogenic process by playing a major role in the generation of mutations in tumor suppressor genes both in germline and somatic cells. It has been demonstrated that the 5-methylcytosine to thymine transition mutation occurs about ten times as frequently as do other transitions.; Because of the difficulties involved in obtaining highly methylated DNA for experimental purposes, the susceptibility of 5-methylcytosine to the damaging effects of UV and ionizing radiation has not been extensively studied. I employed DNA methylase to transfer radioactively labeled methyl groups from S-adenosyl-L-methionine to cytosine residues of poly(dG-dC) in vitro. Using this DNA as a substrate, I characterized UV and ionizing radiation induced damage to 5-methylcytosine and its repair by the DNA repair enzymes, E. coli endonuclease III and calf thymus 5-hydroxymethyluracil-DNA glycosylase. I demonstrated that UV radiation causes the release of intact 5-methylcytosine bases from the DNA backbone leaving apyrimidinic (AP) sites in DNA. This reaction is oxygen dependent, occurs via triplet state excitation of 5-methylcytosine and is probably mediated via free radical formation. In contrast, the exposure of 5-methylcytosine in DNA to oxidative stresses such as ionizing radiation, {dollar}rm Hsb2Osb2,{dollar} and OsO{dollar}sb4{dollar} results in the formation of thymine glycol. This lesion is excised from DNA by E. coli endonuclease III. Finally, to study the mechanism of action of endonuclease III, I demonstrated that the (5S,6R) and (5R,6S) thymidine glycol diastereomers in DNA are differentially repaired.; These results indicated that UV radiation and ionizing radiation caused damage to 5-methylcytosine which is potentially mutagenic to the cell. Both AP sites and thymine glycols are premutagenic because translesion DNA synthesis across either of these lesions will result in an adenine residue opposite the damaged or absent 5-methylcytosine. This will result in a GC to AT transition mutation as well as the loss of a stable methylated site in genomic DNA.