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Comparative Study
. 2008 Jul 22;105(29):10197-202.
doi: 10.1073/pnas.0802816105. Epub 2008 Jul 10.

The Relationship Between dNTP Pool Levels and Mutagenesis in an Escherichia Coli NDP Kinase Mutant

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Free PMC article
Comparative Study

The Relationship Between dNTP Pool Levels and Mutagenesis in an Escherichia Coli NDP Kinase Mutant

Jared Nordman et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Loss of nucleoside diphosphate kinase (Ndk) function in Escherichia coli results in an increased frequency of spontaneous mutation and an imbalance in dNTP pool levels. It is presumed that the imbalance in dNTP pool levels is responsible for the mutator phenotype of an E. coli ndk mutant. A human homologue of Ndk and potential suppressor of tumor metastasis, nm23-H2, can complement the mutagenic phenotype of an E. coli ndk mutant. Here, we show that the antimutagenic property of nm23-H2 in E. coli is independent of dNTP pool levels, indicating that dNTP pool imbalance is not responsible for the mutator phenotype associated with the loss of ndk function. We have identified multiple genetic interactions between ndk and genes involved in the metabolism of dUTP, a potentially mutagenic precursor of thymidine biosynthesis. We show that loss of ndk function is synergistic with a dut-1 mutation and synthetically lethal with the loss of thymidine kinase function. Our results suggest that Ndk prevents the accumulation of dUTP in vivo. Based on these results and biochemical studies of Ndk, we propose that the mutagenic phenotype of an ndk mutant is caused by excess misincorporation of uracil in place of thymidine combined with a defect in the uracil base excision pathway.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Human nm23-H2 does not fully complement an E. coli ndk mutant. (A) Frequency of spontaneous Rif resistance in four to seven independent cultures for each strain tested. Median and interquartile ranges are presented. The median values are 2.5, 38.46, 1.38, and 2.10 for WT, ndk, ndk/pndk, and ndk/pnm23-H2, respectively. (B) Cells of a dnaA(cos) or dnaA(cos) ndk double mutant harboring the indicated plasmid were grown to log phase at 42°C, 10-fold serially diluted, and incubated at 42°C or 30°C for 18 or 24 h, respectively.
Fig. 2.
Fig. 2.
Loss of tdk function is synthetically lethal with loss of ndk function, which is only partially complemented by human nm23-H2. A single representative P1 transduction quantifying the efficiency that an ndk::EZ-Tn5 allele was transduced into a Δtdk mutant harboring the indicated plasmids is shown. A xerD::EZ-Tn5 allele was used as a control for P1 transduction frequencies. * indicates no colonies were detected.
Fig. 3.
Fig. 3.
E. coli NDP kinase genetic and biochemical interactions suggest that ndk could function at the interface between dNTP synthesis and base excision repair. The uracil glycosylase base excision repair pathway (Left) and the pathway of de novo thymidylate biosynthesis pathway (Right) are shown. Arrows with asterisk indicate genetic interactions identified in this study. The Ndk/Ung functional biochemical interaction was identified by Goswami et al. (23). Additionally, Ndk and human Nm23-H2 have been shown to cleave DNA and suggested to do so in a mechanism similar to AP lyases (18). The schematic representation of the uracil BER pathway was adapted from ref. .

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