Roel M. Schaaper

Interactions among the Escherichia coli mutT, mutM, and mutY damage prevention pathways

We have investigated in detail the interactions between the Escherichia coli mutT, mutM, and mutY error-prevention systems. Jointly, these systems protect the cell against the effects of the oxidative stress product, 8-oxoguanine (8-oxoG), a base analog with ambiguous base-pairing properties, pairing with either A or C during DNA synthesis. mutT mutator strains display a specific increase in A.T-->C.G transversions, while mutM and mutY mutator strains show specific G.C-->T.A increases. To study in more detail the in vivo processing of the various mutational intermediates leading to A.T-->C.G and G.C-->T.A transversions, we analyzed defined A.T-->C.G and G.C-->T.A events in strains containing all possible combinations of these mutator alleles. We report three major findings. First, we do not find evidence that the mutT allele significantly increases G.C-->T.A transversions in either mut(+), mutM, mutY or mutMmutY backgrounds. We interpret this result to indicate that incorporation of 8-oxodGTP opposite template C may not be frequent relative to incorporation opposite template A. Second, we show that mutT-induced A.T-->C.G transversions are significantly reduced in strains carrying mutY and mutMmutY deficiencies suggesting that 8-oxoG, when present in DNA, preferentially mispairs with dATP. Third, the mutY and mutMmutY deficiencies also decrease A.T-->C.G transversions in the mutT(+) background, suggesting that, even in the presence of functional MutT protein, A.T-->C.G transversions may still result from 8-oxodGTP misincorporation.

DNA polymerase II as a fidelity factor in chromosomal DNA synthesis in Escherichia coli

Escherichia coli DNA polymerase III holoenzyme (HE) is the main replicase responsible for replication of the bacterial chromosome. E. coli contains four additional polymerases, and it is a relevant question whether these might also contribute to chromosomal replication and its fidelity. Here, we have investigated the role of DNA polymerase II (Pol II) (polB gene product). Mismatch repair‐defective strains containing the polBex1 allele – encoding a polymerase‐proficient but exonucleolytically defective Pol II – displayed a mutator activity for four different chromosomal lac mutational markers. The mutator effect was dependent on the chromosomal orientation of the lacZ gene. The results indicate that Pol II plays a role in chromosomal replication and that its role is not equal in leading‐ versus lagging‐strand replication. In particular, the role of Pol II appeared larger in the lagging strand. When combined with dnaQ or dnaE mutator alleles, polBex1 showed strong, near multiplicative effects. The results fit a model in which Pol II acts as proofreader for HE‐produced misinsertion errors. A second role of Pol II is to protect mismatched 3′ termini against the mutagenic action of polymerase IV (dinB product). Overall, Pol II may be considered a main player in the polymerase trafficking at the replication fork.

The θ Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the ε Proofreading Subunit

The DNA polymerase III (Pol III) holoenzyme (HE) is the major chromosomal replication enzyme in Escherichia coli (19, 22, 30, 31). The enzyme is composed of 17 subunits, 10 of which are distinct (19, 31). HE contains two polymerase core molecules, each consisting of an α, ɛ, and θ subunit arranged in the linear order α-ɛ-θ. The two cores are connected through a dimer of the τ subunit, establishing the basic arrangement of a dimeric polymerase that simultaneously replicates the leading and lagging strands (14). In addition, the HE contains, for each core, a β-clamp (β2) tethering the polymerase to the DNA to ensure high processivity and the five-subunit γ-complex (γδδ′χψ) responsible for loading and unloading the β-clamp. The precise functioning of the HE complex in chromosomal replication is under active investigation (5, 24, 26, 31). Within the Pol III core, α (the dnaE gene product) is the DNA polymerase, while ɛ (the dnaQ gene product) is the 3′→5′ proofreading exonuclease. The function of θ (the holE gene product), which is tightly bound to ɛ, is unclear. Genetic analysis of the Pol III core constituents has provided insight into the role of its constituents. For example, dnaE(Ts) mutants, encoding temperature-sensitive polymerase subunits, are conditionally lethal, as expected in view of the essential nature of the Pol III replication function. Several dnaE mutants display mutator or antimutator effects (9, 28), indicating the important fidelity role of this enzyme. Many dnaQ mutants exhibit strong mutator phenotypes, indicating the importance of the 3′-exonuclease activity for replication fidelity (45). Deletion mutants of dnaQ (23, 27) or mutants lacking the domain necessary for interaction with the polymerase (46) have been generated, but these mutants proved essentially inviable unless accompanied by a suppressing mutation in dnaE. Based on these studies, ɛ is assigned at least two functions: a fidelity function through its 3′-exonuclease activity and a structural function based on its tight, and presumably stabilizing, interaction with the polymerase (23, 27, 45, 46). In contrast, the role of the θ subunit of the Pol III core is unknown. Loss of θ (ΔholE) results in healthy cells with no morphology changes and little or no change in mutant frequencies (42). Based on these studies, it was suggested that θ is not necessary or important for proper functioning of the Pol III core. θ does not affect DNA synthesis by α or α-ɛ (43); however, gel filtration (44), coexpression (1), and yeast two-hybrid experiments (18) have demonstrated a tight interaction between θ and ɛ, but none between θ and α. Interestingly, θ was shown to moderately affect the 3′→5′ proofreading exonuclease activity, as addition of θ to an exonuclease assay measuring the removal of a G · T mispair increased ɛ-mediated excision of the terminal T residue by about 2.5-fold (44). The above findings suggest that θ, while not essential, could play a role in DNA replication and its fidelity, presumably indirectly through its interaction with the ɛ subunit. The precise nature of this interaction is being pursued structurally by both nuclear magnetic resonance (NMR) and crystallography studies. Structures of both ɛ and θ have been reported (6, 15, 20), as well as the ɛ-θ interaction surface on ɛ (7). Here, we report on a series of genetic experiments on the ɛ-θ interaction. Specifically, we have studied (i) in greater detail, the possible mutator effect resulting from a holE deletion, (ii) the effect of the holE deletion on dnaQ mutator mutants, and (iii) the effect of θ on the α-ɛ interaction as measured by yeast two- and three-hybrid assays. The results suggest that θ may be a stabilizing factor for the ɛ subunit, which has been shown to be intrinsically unstable (11).

Reaction mechanism of the ε subunit of E. coli DNA polymerase III: Insights into active site metal coordination and catalytically significant residues

The 28 kDa epsilon subunit of Escherichia coli DNA polymerase III is the exonucleotidic proofreader responsible for editing polymerase insertion errors. Here, we study the mechanism by which epsilon carries out the exonuclease activity. We performed quantum mechanics/molecular mechanics calculations on the N-terminal domain containing the exonuclease activity. Both the free-epsilon and a complex epsilon bound to a theta homologue (HOT) were studied. For the epsilon-HOT complex Mg(2+) or Mn(2+) were investigated as the essential divalent metal cofactors, while only Mg(2+) was used for free-epsilon. In all calculations a water molecule bound to the catalytic metal acts as the nucleophile for hydrolysis of the phosphate bond. Initially, a direct proton transfer to H162 is observed. Subsequently, the nucleophilic attack takes place followed by a second proton transfer to E14. Our results show that the reaction catalyzed with Mn(2+) is faster than that with Mg(2+), in agreement with experiment. In addition, the epsilon-HOT complex shows a slightly lower energy barrier compared to free-epsilon. In all cases the catalytic metal is observed to be pentacoordinated. Charge and frontier orbital analyses suggest that charge transfer may stabilize the pentacoordination. Energy decomposition analysis to study the contribution of each residue to catalysis suggests that there are several important residues. Among these, H98, D103, D129, and D146 have been implicated in catalysis by mutagenesis studies. Some of these residues were found to be structurally conserved on human TREX1, the exonuclease domains from E. coli DNA-Pol I, and the DNA polymerase of bacteriophage RB69.

YcbX and yiiM, two novel determinants for resistance of Escherichia coli to N‐hydroxylated base analogues

We have shown previously that lack of molybdenum cofactor (MoCo) in Escherichia coli leads to hypersensitivity to the mutagenic and toxic effects of N‐hydroxylated base analogues, such as 6‐N‐hydroxylaminopurine (HAP). However, the nature of the MoCo‐dependent mechanism is unknown, as inactivation of all known and putative E. coli molybdoenzymes does not produce any sensitivity. Presently, we report on the isolation and characterization of two novel HAP‐hypersensitive mutants carrying defects in the ycbX or yiiM open reading frames. Genetic analysis suggests that the two genes operate within the MoCo‐dependent pathway. In the absence of the ycbX‐ and yiiM‐dependent pathways, biotin sulfoxide reductase plays also a role in the detoxification pathway. YcbX and YiiM are hypothetical members of the MOSC protein superfamily, which contain the C‐terminal domain (MOSC) of the eukaryotic MoCo sulphurases. However, deletion of ycbX or yiiM did not affect the activity of human xanthine dehydrogenase expressed in E. coli, suggesting that the role of YcbX and YiiM proteins is not related to MoCo sulphuration. Instead, YcbX and YiiM may represent novel MoCo‐dependent enzymatic activities. We also demonstrate that the MoCo/YcbX/YiiM‐dependent detoxification of HAP proceeds by reduction to adenine.

Multiple antimutagenesis mechanisms affect mutagenic activity and specificity of the base analog 6-N-hydroxylaminopurine in bacteria and yeast.

Base analog 6-N-hydroxylaminopurine is a potent mutagen in variety of prokaryotic and eukaryotic organisms. In the review, we discuss recent results of the studies of HAP mutagenic activity, genetic control and specificity in bacteria and yeast with the emphasis to the mechanisms protecting living cells from mutagenic and toxic effects of this base analog.

Mutator Phenotype Resulting from DNA Polymerase IV Overproduction in Escherichia coli: Preferential Mutagenesis on the Lagging Strand

We investigated the mutator effect resulting from overproduction of Escherichia coli DNA polymerase IV. Using lac mutational targets in the two possible orientations on the chromosome, we observed preferential mutagenesis during lagging strand synthesis. The mutator activity likely results from extension of mismatches produced by polymerase III holoenzyme.

Asymmetry of frameshift mutagenesis during leading and lagging-strand replication in Escherichia coli.

Mutations in DNA, including frameshifts, may arise during DNA replication as a result of mistakes made by the DNA polymerase in copying the DNA template strands. In our efforts to better understand the factors that contribute to the accuracy of DNA replication, we have investigated whether frameshift mutations on the Escherichia coli chromosome occur differentially within the leading and lagging-strands of replication. The experimental system involves measurement of the reversion frequency for several defined lac frameshift alleles in pairs of strains in which the lac target is oriented in the two possible directions relative to the origin of chromosomal replication. Within these pairs any defined lac sequence will be subject to leading-strand replication in one orientation and to lagging-strand replication in the other. Fidelity differences between the two modes of replication can be observed as a differential lac reversion between the two strains. Our results, obtained with a series of lac alleles in a mismatch-repair-defective background, indicate that for at least some of the alleles there is indeed a difference in the fidelity of replication between the two modes of replication.

Hypersensitivity of Escherichia coli Δ(uvrB-bio) Mutants to 6-Hydroxylaminopurine and Other Base Analogs Is Due to a Defect in Molybdenum Cofactor Biosynthesis

We have shown previously that Escherichia coli and Salmonella enterica serovar Typhimurium strains carrying a deletion of the uvrB-bio region are hypersensitive to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and related base analogs. This sensitivity is not due to the uvrB excision repair defect associated with this deletion because a uvrB point mutation or a uvrA deficiency does not cause hypersensitivity. In the present work, we have investigated which gene(s) within the deleted region may be responsible for this effect. Using independent approaches, we isolated both a point mutation and a transposon insertion in the moeA gene, which is located in the region covered by the deletion, that conferred HAP sensitivity equal to that conferred by the uvrB-bio deletion. The moeAB operon provides one of a large number of genes responsible for biosynthesis of the molybdenum cofactor. Defects in other genes in the same pathway, such as moa or mod, also lead to the same HAP-hypersensitive phenotype. We propose that the molybdenum cofactor is required as a cofactor for an as yet unidentified enzyme (or enzymes) that acts to inactivate HAP and other related compounds.

Role of DNA Polymerase IV in Escherichia coli SOS Mutator Activity

Constitutive expression of the SOS regulon in Escherichia coli recA730 strains leads to a mutator phenotype (SOS mutator) that is dependent on DNA polymerase V (umuDC gene product). Here we show that a significant fraction of this effect also requires DNA polymerase IV (dinB gene product).