Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent

Highlights: • The intracellular free iron in E.coli hemH appear to be double that in wt strain. • Increased Fe(II) and AlkB concentrations result in decreased MMS-induced mutations. • Dealkylation of dNTPs takes place in the presence of Fe(II) and not requires AlkB. - Abstract: An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH⁺ strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg⁺ reversion induction after λ > 320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O₂) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg⁺ revertants in AB1157 alkB⁺hemH^–/pMW1 strain was 40 and 26% reduced comparing to the alkB⁺hemH^– and alkB⁺hemH⁺/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ > 320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg⁺ reversion was observed in irradiated and MMS-treated hemH^–alkB^– cells comparing to the hemH⁺alkB^– strain. Also, the level of Arg⁺ revertants in the irradiated and MMS treated hemH^– alkB^– mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH^– intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs