Unique Structural Features in DNA Polymerase IV Enable Efficient Bypass of the N 2 Adduct Induced by the Nitrofurazone Antibiotic

Abstract

The reduction in the efficacy of therapeutic antibiotics represents a global problem of increasing intensity and concern. Nitrofuran antibiotics act primarily through the formation of covalent adducts at the N(2) atom of the deoxyguanosine nucleotide in genomic DNA. These adducts inhibit replicative DNA polymerases (dPols), leading to the death of the prokaryote. N(2)-furfuryl-deoxyguanosine (fdG) represents a stable structural analog of the nitrofuran-induced adducts. Unlike other known dPols, DNA polymerase IV (PolIV) from E. coli can bypass the fdG adduct accurately with high catalytic efficiency. This property of PolIV is central to its role in reducing the sensitivity of E. coli toward nitrofuran antibiotics such as nitrofurazone (NFZ). We present the mechanism used by PolIV to bypass NFZ-induced adducts and thus improve viability of E. coli in the presence of NFZ. Our results can be used to develop specific inhibitors of PolIV that may potentiate the activity of nitrofuran antibiotics.

Noncognate DNA damage prevents the formation of the active conformation of the Y‐family DNA polymerases DinB and DNA polymerase κ

“…Only rather subtle dNTP‐induced conformational changes in Y‐family DNA Pols have been observed. Indeed, the difference in rmsd in the crystal structures of DinB ternary complexes with a correct or an incorrect incoming nucleotide is 0.35–0.43 Å , which is similar (0.4 Å) to what is found in ternary complex structures of DinB bound to undamaged DNA or DNA containing N 2 fG . Dpo4 undergoes an apparent movement of domains upon binding to DNA, but very little or no change in fluorescence resonance energy transfer efficiency is observed upon formation of a ternary complex .…”

Reactive Oxygen Species Play an Important Role in the Bactericidal Activity of Quinolone Antibiotics

“…Foti and colleagues showed that incorporation of the oxidized nucleotide poolespecially 8oxodGTP (8odGTP)-into the genome by PolIV contributes substantially to the deleterious effects of ROS on cellular physiology. [7] PolIV has significant ability to incorporate 8odGTP opposite dA but not dC,e ven at high concentrations of 8odGTP ( Figure 1). [5b,6] The8 odGTP incorporation activity of PolIV is in addition to previously observed roles in stress-induced mutagenesis or translesion synthesis that aid survival of the cell.…”

Point mutations in Escherichia coli DNA pol V that confer resistance to non-cognate DNA damage also alter protein–protein interactions

“…Using hydroxylamine mutagenesis, we identified variants of E. coli UmuC (Fig. 1) that conferred resistance to nitrofurazone (NFZ), which is thought to cause the N 2 -furfuryl-dG lesion, considered a cognate lesion of DinB [41,43] but a non-cognate lesion of UmuC (Fig. 1).…”

“…whereas UmuC variants H282P and T412I do not N 2 -furfuryl-dG is thought to be the lesion resulting from treatment with NFZ and is considered to be a cognate lesion of DinB [41][42][43]. Agents that form N 2 -dG adducts can also form N 6 -dA adducts.…”

“…Pol V also bypasses C 8 -dG-acetylaminofluorine inaccurately, by adding dA opposite the lesion instead of dC [20,39,40]. N 2 -furfuryl-dG, thought to be formed upon exposure to nitrofurazone (NFZ), is considered to be the cognate lesion of DinB [41][42][43], as DinB bypasses this lesion, as well as other N 2 -dG adducts such as N 2 -benzo[a]pyrene-dG [44,45] and N 2 -(1-carboxyethyl)-dG [46] accurately by adding dC opposite the lesion.…”