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5-methylcytosine methylation damage; DNA base excision repair; N-4,5-dimethylcytosine; epigenetics
Abstract:
Cytosine (C) in DNA is often modified to 5-methylcytosine (m(5)C) to execute important cellular functions. Despite the significance of m(5)C for epigenetic regulation in mammals, damage to m(5)C has received little attention. For instance, almost no studies exist on erroneous methylation of m(5)C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m(5)C into N-4,5-dimethylcytosine (m(N4,5)C) inDNA, m(N4,5)C is probably present in vivo. We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the Escherichia coli Nei and Fpg proteins at m(N4,5)C residues in vitro. The activity of Nei was highest opposite cognate guanine followed by adenine, thymine (T) and C. Fpg-complemented Nei by exhibiting the highest activity opposite C followed by lower activity opposite T. To our knowledge, this is the first description of a repair enzyme activity at a further methylated m(5)C in DNA, as well as the first alkylated base allocated as a Nei or Fpg substrate. Based on our observed high sensitivity to nuclease S1 digestion, we suggest that m(N4,5)C occurs as a disturbing lesion inDNA and that Nei may serve as a major DNA glycosylase in E. coli to initiate its repair.
