Hains, K. | Search bioRxiv

bioRxiv2026

Introduction of CG methylation in E. coli induces mutagenesis at AT base pairs

In many eukaryotic species, DNA is methylated at the 5 position of cytosine to form 5mC, predominantly within CG dinucleotides. Despite being conserved since the dawn of eukaryotic life, 5mC is often lost from individual lineages, suggesting that it may have detrimental effects. One such effect is genotoxicity, through the effect of 5mC on the process of cytosine deamination and its repair. Additionally, enzymes that introduce 5mC (DNA methyltransferases, DNMTs) can also damage DNA through alkylation and oxidative stress, but how these genotoxic effects combine to influence mutagenesis is unclear. To investigate how mutagenesis changes upon methylation of CG dinucleotides we introduced high levels of CG methylation into the bacteria E. coli. 5mC induction increased mutation at CG dinucleotides consistent with increased C to T mutations. We also discovered that 5mC induction led to increased mutations at AT base pairs, specifically in the absence of the alkylation repair enzyme AlkB. This effect was specific to certain E. coli strains and was not dependent on the DNA repair enzyme RecA, so its exact mechanism remains unclear. Together, our work highlights multiple mutagenic consequences of DNMT expression, which might act as selective pressures for organisms to lose 5mC across evolution.

molecular biology↗

bioRxiv2024

Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage

DNA methylation at the 5 position of cytosine (5mC) is an ancient epigenetic mark in eukaryotes. The levels of total 5mC vary enormously between different species, and the DNA methyltransferases that introduce 5mC have been repeatedly lost in several independent lineages. DNA methyltransferases are a threat to genomic stability due to the increased mutagenicity of 5mC bases and the propensity of DNA methyltransferases themselves to introduce DNA alkylation damage as an off-target effect. However, whether alkylation damage explains why 5mC is frequently lost in evolution is unclear. Here we tested the fitness consequences of DNA methyltransferase-induced alkylation damage by introducing a eukaryotic-like 5mC system into E. coli. We showed that introducing 5mC genome-wide leads to increased sensitivity to alkylating agents, which is strongly enhanced by removal of the 3mC repair enzyme AlkB. Unexpectedly, we discovered that 5mC introduction led to increased sensitivity to oxidative stress. We showed that this is due to increased formation of reactive oxygen in the presence of 5mC. We determined that reactive oxygen species led to non-enzymatic oxidation of 5mC, producing modified cytosines such as 5fC that are recognised as DNA base damage in E. coli. Overall, our work identifies increased sensitivity to oxidative stress, as well as alkylating agents, as a negative consequence of genome-wide 5mC. Oxidative stress is frequently encountered by organisms in their environment, thus offering a plausible reason for total loss of 5mC in some species.

molecular biology↗