Nucleoside Diphosphate Kinase Escalates A-to-C Mutations in MutT-Deficient Strains of Escherichia coli

Abstract

The chemical integrity of the nucleotide pool and its homeostasis are crucial for genome stability. Nucleoside diphosphate kinase (NDK) is a crucial enzyme that carries out reversible conversions from nucleoside diphosphate (NDP) to nucleoside triphosphate (NTP) and deoxynucleoside diphosphate (dNDP) to deoxynucleoside triphosphate (dNTP). Guanosine nucleotides (GDP, GTP, dGDP, and dGTP) are highly susceptible to oxidative damage to 8-oxo-GDP (8-O-GDP), 8-O-dGTP, 8-O-GTP, and 8-O-dGTP. MutT proteins in cells hydrolyze 8-O-GTP to 8-O-GMP or 8-O-dGTP to 8-O-dGMP to avoid its incorporation in nucleic acids. In Escherichia coli, 8-O-dGTP is also known to be hydrolyzed by RibA (GTP cyclohydrolase II). In this study, we show that E. coli NDK catalyzes the conversion of 8-O-dGDP to 8-O-dGTP or vice versa. However, the rate of NDK-mediated phosphorylation of 8-O-dGDP to 8-O-dGTP is about thrice as efficient as the rate of dephosphorylation of 8-O-dGTP to 8-O-dGDP, suggesting an additive role of NDK in net production of 8-O-dGTP in cells. Consistent with this observation, the depletion of NDK (Δndk) in E. coli ΔmutT or ΔmutT ΔribA strains results in a decrease of A-to-C mutations. These observations suggest that NDK contributes to the physiological load of MutT in E. coliIMPORTANCE Nucleoside diphosphate kinase (NDK), a ubiquitous enzyme, is known for its critical role in homeostasis of cellular nucleotide pools. However, NDK has now emerged as a molecule with pleiotropic effects in DNA repair, protein phosphorylation, gene expression, tumor metastasis, development, and pathogen virulence and persistence inside the host. In this study, we reveal an unexpected role of NDK in genome instability because of its activity in converting 8-O-dGDP to 8-O-dGTP. This observation has important consequences in escalating A-to-C mutations in Escherichia coli The severity of NDK in enhancing these mutations may be higher in the organisms challenged with high oxidative stress, which promotes 8-O-dGDP/8-O-dGTP production.

Keywords: 8-oxo-dGTP (8-O-dGTP); MutT; NDK; dNTP; mutation rate and mutation frequency.

FIG 1

Purification of EcoNDK from the E. coli mutT null strain and activity analysis on 8-O-dGTP. (A) Representative 15% SDS-PAGE gel showing the purity and migration of the purified EcoNDK. M stands for the protein size markers. (B) Chromatograms indicate NDK-catalyzed reaction on 8-O-dGTP. Reaction analytes were separated on a DNAPac column through high-performance liquid chromatography (HPLC), using a gradient of 1 M LiCl (0% to 40%). The retention times of different analytes are shown on the x axis and the corresponding peak intensities (given in arbitrary units) are shown on the y axis. (i) Control. Shown is the migration profile of ADP and 8-O-dGTP. (ii) Reaction in the presence of NDK. Shown is the emergence of ATP and 8-O-dGDP peaks due to NDK-mediated transfer of terminal phosphate from 8-O-dGTP to ADP.

FIG 2

Summary of the reaction rate analysis.

FIG 3

Mutation frequency analyses. (A) Mutation frequency analysis of MG1655 ΔmutT::kan strain harboring a plasmid (pBAD or pBAD_Eco_mutT or pBAD_Eco_ndk [refer to Table S2]). Mutation frequencies (using 4 replicates for each strain) were determined by dividing the number of colonies that appeared on the LB plus Amp plate containing rifampin by the corresponding viable counts obtained on the LB plus Amp plate. (B) The expression of plasmid-borne EcoMutT or EcoNDK in the MG1655 ΔmutT::kan strain was verified under the experimental conditions.

FIG 4

Mutation frequency analyses. (A) Analysis of A-to-C (or T-to-G) mutation frequency of the CC101 ΔmutT::kan strain harboring the pBAD vector or its derivatives expressing EcoMutT or EcoNDK (refer to Table S3). Reversion frequencies (using 9 or 10 replicates for each strain) were calculated by dividing the number of colonies that appeared on the minimal medium Amp plate containing lactose by the corresponding CFU on the minimal medium Amp plate containing glucose. (B) The expression of plasmid-borne EcoMutT or EcoNDK in the CC101 ΔmutT::kan strain was confirmed during the assay conditions.

FIG 5

Comparisons of Lac⁺ reversion frequencies between CC101 ΔmutT and CC101 ΔmutT Δndk strains. Panels A and B show two independent data sets. Reversion frequencies were calculated by dividing the number of spontaneous revertants by the corresponding viable counts. The plotted median and confidence intervals are calculated with ≥95% of the confidence of the coefficient, using 10 replicates for each strain (refer to Table S4).

FIG 6

Comparisons between CC101 ΔmutT ΔribA and CC101 ΔmutT ΔribA Δndk strains. Panels A and B represent two independent comparisons of Lac⁺ reversion frequencies. Reversion frequencies were obtained by dividing the number of Lac⁺ revertants by the respective viable counts. (C) Lac⁺ reversion rates were estimated as described in Materials and Methods. Each comparative plot represents median and ≥95% confidence intervals estimated using 10 or 11 replicates of each strain (refer to Tables S5 and S6).