| dc.description.abstract | 5-Methylcytosine (m5C) replaces cytosine (C) in DNA in prokaryotic and eukaryotic cells to execute a number of important cellular functions, but damage to m5C have received little attention. For instance, almost no studies exist on erroneous methylation of m5C by alkylating agents to double and triple methylated bases. Due to chemical evidence, and because many prokaryotes express methylases able to convert m5C into the double methylated N4,5-dimethylcytosine (mN4,5C) in DNA, this base lesion is likely to be present in cellular DNA.
In this study, we used DNA with one mN4,5C residue incorporated at a specific site where mN4,5C was placed opposite G, A, C or T (called mN4,5C:G-DNA, mN4,5C:A-DNA, mN4,5C:C-DNA and mN4,5C:T-DNA, respectively. We set up several experiments to find putative repair activity for mN4,5C using different E. coli and human glycosylases, major initiators of the base excision repair (BER) pathway. We found the highest repair activity for mN4,5C:C-DNA and mN4,5C:G-DNA by the two Escherichia coli DNA glycosylases Fpg and Nei, respectively. To confirm this repair activity, endonuclease IV and T4-polynucleotide kinase were employed to define and process the 3´-end products following Fpg and Nei-mediated incision of mN4,5C:C-DNA and mN4,5C:G-DNA, respectively. In contrast, several other E. coli DNA glycosylases like Ung and Mug and the human enzyme hUNG did not show repair activity at employed conditions. To our knowledge, this is the first report describing a repair activity for a further methylated m5C in DNA as well as the first alkylated base allocated to Fpg and Nei as substrate. | nb_NO |
