Mutational consequences of dNTP pool imbalances in E. coli

Abstract

The accuracy of DNA synthesis depends on the accuracy of the polymerase as well as the quality and concentration(s) of the available 5'-deoxynucleoside-triphosphate DNA precursors (dNTPs). The relationships between dNTPs and error rates have been studied in vitro, but only limited insights exist into these correlations during in vivo replication. We have investigated this issue in the bacterium Escherichia coli by analyzing the mutational properties of dcd and ndk strains. These strains, defective in dCTP deaminase and nucleoside diphosphate kinase, respectively, are characterized by both disturbances of dNTP pools and a mutator phenotype. ndk strains have been studied before, but were included in this study, as controversies exist regarding the source of its mutator phenotype. We show that dcd strains suffer from increased intracellular levels of dCTP (4-fold) and reduced levels of dGTP (2-fold), while displaying, as measured using a set of lacZ reversion markers in a mismatch-repair defective (mutL) background, a strong mutator effect for G·C→T·A and A·T→T·A transversions (27- and 42-fold enhancement, respectively). In contrast, ndk strains possess a lowered dATP level (4-fold) and modestly enhanced dCTP level (2-fold), while its mutator effect is specific for just the A·T→T·A transversions. The two strains also display differential mutability for rifampicin-resistant mutants. Overall, our analysis reveals for both strains a satisfactory correlation between dNTP pool alterations and the replication error rates, and also suggests that a minimal explanation for the ndk mutator does not require assumptions beyond the predicted effect of the dNTP pools.

Fig. 1

Pathways for dNTP biosynthesis in E. coli. RNR = ribonucleotide reductase, Ndk = Nucleoside diphosphate kinase, Dcd = dCTP deaminase, Dut = dUTPase, ThyA = Thymidylate synthase, Tmk = dTMP kinase. The pathway in parentheses along with the stippled paths indicates the minor pathway (~ 20%) for dUMP (and dTTP) synthesis. The major pathway for dTTP synthesis is via dCTP and dCTP deaminase.

Fig. 2

Predicted effects of measured dNTP changes in the E. coli dcd mutant on replication errors. Shown are the twelve possible mispairing errors for the six different base substitutions that can be scored in the lacZ reversion system [29] as well as the (next) dNTP to be inserted immediately following the mispair. The format for the mispairs is: template base·(wrong base/correct base). In red, we denote dCTP or dGTP changes that are predicted to promote the indicated errors, either by favoring misinsertion or by promoting mispair extension; in blue, dCTP or dGTP changes that would disfavor the errors. The wild-type DNA sequence at the site of lac reversion is 5′-AAT GAG AGT-3′, while in strains CC101 through CC106 the GAG codon (encoding the essential glutamic acid residue) is replaced by TAG, GGG, CAG, GCG, GTG, or AAG, respectively [29]. Only the single-base change to GAG will restore the Lac⁺ phenotype. The preferred mispairings for four of the six base-pair substitutions are bolded and boxed. See text for further details.

Fig. 3

Predicted effects of measured dNTP changes in the E. coli ndk mutant on replication errors. Shown are the twelve possible mispairing errors for the six different base substitutions that can be scored in the lacZ reversion system [29] as well as the (next) dNTP to be inserted immediately following the mispair. In red, we denote dATP or dCTP changes that are predicted to promote the indicated errors, either by favoring misinsertion or by promoting mispair extension; in blue, dATP or dCTP changes that would disfavor the errors. See Legend to Fig. 2 and text for further details.