The escherichia coli alkA gene is activated to alleviate mutagenesis by an oxidized deoxynucleoside

Abstract

The cellular methyl donor S-adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (m³A), which in <i>Escherichia coli</i> is removed by m³A-DNA glycosylase I (Tag) and II (AlkA). The <i>tag</i> gene is constitutively expressed, while <i>alkA</i> is induced by sub-lethal concentrations of methylating agents. We previously found that AlkA exhibits activity for the reactive oxygen-induced thymine (T) lesion 5-formyluracil (fU) <i>in vitro</i>. Here, we provide evidence for AlkA involvement in the repair of oxidized bases by showing that the adenine (A) ⋅ T → guanine (G) ⋅ cytosine (C) mutation rate increased 10-fold in <i>E. coli</i> wild-type and <i>alkA</i>– cells exposed to 0.1 mM 5-formyl-2′-deoxyuridine (fdU) compared to a wild-type specific reduction of the mutation rate at 0.2 mM fdU, which correlated with <i>alkA</i> gene induction. G⋅C → A⋅T alleviation occurred without <i>alkA</i> induction (at 0.1 mM fdU), correlating with a much higher AlkA efficiency for fU opposite to G than for that to A. The common keto form of fU is the AlkA substrate. Mispairing with G by ionized fU is favored by its exclusion from the AlkA active site.