Cytosine (C) in DNA is often modified to 5-methylcytosine (m⁵C) to execute important cellular functions. Despite the significance of m⁵C for epigenetic regulation in mammals, damage to m⁵C has received little attention. For instance, almost no studies exist on erroneous methylation of m⁵C by alkylating agents to doubly or triply methylated bases. Owing to chemical evidence, and because many prokaryotes express methyltransferases able to convert m⁵C into <i>N</i>⁴,5-dimethylcytosine (m<i>^N</i>^4,5C) in DNA, m<i>^N</i>^4,5C is probably present <i>in vivo</i>. We screened a series of glycosylases from prokaryotic to human and found significant DNA incision activity of the <i>Escherichia coli</i> Nei and Fpg proteins at m<i>^N</i>^4,5C residues <i>in vitro</i>. 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 m⁵C 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<i>^N</i>^4,5C occurs as a disturbing lesion in DNA and that Nei may serve as a major DNA glycosylase in <i>E. coli</i> to initiate its repair.This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.