Excision of the doubly methylated base N ⁴ ,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins

Abstract

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

Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

“…This resulted in an 11-nt incision product with an active glycosylase enzyme (see Figure 1A ). The DNA at the defined site, which contains m N 4 C and m N 4,5 C, was prepared as described previously by Alexeeva et al (2018) . The ss polydeoxyribonucleotide [Cy3] 5′-CCCTCGAT GTA[U]CATGGATCCGATCGATCC-3′ (Fw 30 nt; 11 nt incision product) containing uracil (U) at the specific site was annealed to equimolar amounts of the Rev strand with G opposite U, and used as a positive control substrate [for active enzyme, Fpg AP lyase activity, after incubation with uracil-DNA glycosylase (Ung)].…”

“…The Nakahara et al report is compounded by our recent discovery that the E. coli Fpg exhibits significant activity where N 4 ,5-dimethylcytosine (m N 4,5 C) placed opposite C or T is removed from DNA in vitro ( Alexeeva et al, 2018 ). The report and our discovery urged us to investigate in detail the ability of Fpg, and therefore the base excision repair (BER) pathway, to incise and repair C, including where methylated C is opposite C. We found that the enzyme exhibited activity for C⋅C.…”

Unique structural features of mammalian NEIL2 DNA glycosylase prime its activity for diverse DNA substrates and environments

“…The strong AP site activity on both single and double-stranded DNA suggests that either the abasic site is a true substrate for NEIL2, the favored base modification substrate is as of yet unidentified, or sequence-context dependence for a particular lesion may exist. Indeed, it was recently shown that both EcoNei and Fpg will excise N4,5dimethylcytosine while leaving 5-methylcytosine and 4-methylcytosine uncut (42). Additionally, both NEIL1 and NEIL2 were shown to function in TDG-mediated turnover of 5hmC(11) at sites of TET dependent DNA demethylation while 5hmC accumulated at sites of DNA damage (43) and thus exhibit the ability to substitute for APE1 in the demethylation pathway.…”

Epigenetics: the first 25 centuries

“…How are nucleic acid modifications introduced, how are they recognized or targeted, and to what extent are they heritable?-these are all burning issues in epigenetics at the moment. In this issue, Klimasˇauskas [2] describes studies with the doubly methylated base N 4 ,5-dimethylcytosine using enzymes from Escherichia coli, while the potential for epigenetic modifications to influence alternative splicing is reviewed by Koziol [3]. Gene-specific editing in vitro and in vivo is a hot topic, and the different methodologies available are compared by Rots [4].…”