DNA glycosylase activities for N4,5-dimethylcytosine
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Although the epigenetic DNA base 5-methylcytosine (m5C) has an important role in cellular functions, damaging chemical alterations to m5C have been little studied. For example, while much knowledge exists on erroneous methylation of the four common bases in DNA, almost none studies have been conducted on methylation damage to m5C resulting in double and triple methylated bases. Certain methylases can convert m5C into N4,5-dimethylcytosine (mN4,5C) in DNA in vitro, and there is a possibility of the presence of mN4,5C in vivo. We investigated the ability of various DNA glycosylases to process DNA containing mN4,5C at a specific site, and we report that Escherichia coli Fpg protein and endonuclease VIII (Nei) exhibit activity for mN4,5C in DNA in vitro. Fpg removes mN4,5C most efficiently opposite non-cognate C followed by T, while no activity was detected opposite A and cognate G. In contrast, Nei incises at mN4,5C in DNA most efficiently opposite cognate G followed by A and T, whereas almost no activity was detected opposite C. Nei and Fpg thus seem to complement each other in the repair of mN4,5C in DNA. Plasmids containing mN4,5C placed opposite G, C, A and T should separately be transformed into E. coli wild-type, fpg‒, nei‒ and fpg‒ nei‒ cells to study the in vivo consequences of these repair functions. Our findings describe for the first time the repair of a further methylated epigenetic base in DNA.
Master's thesis in Biological chemistry