DNA METHYLATION AND CHEMICAL CARCINOGENESIS

Abstract

The aim of this project is to study the the structure and biological consequences of DNA containing 6-O-methylguaninc (meG). Synthetic oligonucleotides containing meG were used in the in vitro experiments. The structure of meG containing DNA can be deduced by MaeII digestion of the oligomers. The effect of meG's presence on two systems were evaluated by effect of prokaryotic restriction endonucleases and methylation by human DNA (cytosine-5-)-methyltransferase (DNA MTase). The perturbation produced by presence of meG on the DNA can be detected by cleavage al the site of lesion by restriction endonuclease Maell. In addition, the perturbation can affect the cleavage of DNA by the restriction enzyme Dral which nicks several base-pairs away (either 3' or 5') from the lesion. The possible mechanism and the significance of these striking differences in the rate of cleavage of restriction enzyme on meG containing DNA were discussed. We have chosen DNA methylation as a system to study the effect of the presence of meG in DNA because DNA methylation is believed to control gene expression. Duplexes containing meG·C base pair showed enhanced de novo methylation by the purified human DNA MTase from CEM cells. Subsequent introduction of hemimethylated sites at various positions on the duplexes were used to study the maintenance methylation by the DNA MTase. This revealed striking differences in the rate, amount, and site or methylation, which arc dependent on the position of the hemimethylated site in the duplexes. The possible mechanisms through which DNA MTase acts are discussed. The data from the two systems suggested that the residue in DNA has other genetic effects apart from its miscoding behavior, and these effects could be immediate since it is not necessary to create point mutation by DNA replication. The ability of the human DNA MTase to recognise abnormal bases prompted further interest in understanding the molecular structure and function of the protein. We have purified the human MTase which has been cloned and expressed in E.coli. Similar to the endogenous protein that has been purified from the CEM cells, the recombinant human DNA MTase prefers hemimethylated DNA to nonmethylated DNA as their substrate. The recombinant human DNA MTase is also able to recognise mispaired and meG containing DNA and methylates them differently as compared to DNA containing normal base. In addition, the recombinant DNA MTase was found to bind selectively to A/T rich DNA. The significance of these results, particularly in relation to gene expression and carcinogenesis were discussed.