Effect of episomally encoded DNA polymerases on chemically induced mutagenesis at the hisG46 target in Ames test.

BACKGROUND: The standard Ames test strains owe their high sensitivity to chemical and physical mutagens to the episomal Y-family DNA polymerase RI encoded by the mucAB operon. The S. typhimurium test strains carry also another related samAB operon on a 60-kDa cryptic plasmid. In contrast to the chromosomally encoded Y-family DNA polymerases V and IV, these plasmid born polymerase genes have no direct counterpart in mammalian cells. By replicating damaged templates, DNA polymerases play a central role in mutagenesis and genome stability. It is therefore imperative to investigate their specificity to understand differences in mutagenesis between the prokaryotic versus eukaryotic (mammalian) systems. To this end we have isolated and separately expressed the DNA polymerase subunits encoded by the mucAB and samAB operons. After demonstrating how these enzymes control chemical and UV mutagenesis at the standard hisD3052 and hisG428 Ames test targets, we are now adding the third Ames test target hisG46 to the trilogy. RESULTS: Four new Ames tester strains based on the hisG46 target have been constructed expressing the activated DNA polymerase MucA' and SamA' accessory subunits combined with the MucB and SamB catalytical subunits under the control of lac promoter. These polymerase assemblies were substituted for the endogenous PolRI, PolV and SamAB polymerases present in the standard TA100 strain and tested for their abilities to promote chemically induced mutagenesis. SamA' + SamB has been able to promote mutagenesis induced by AF-2 and 1,8-DNP to higher extent than SamA' + MucB. The MucA' + MucB (PolRI*) more efficiently promoted MMS as well as spontaneous mutagenesis than its wild type counterpart but was less efficient for other mutagens including AFB1. Strikingly azide mutagenesis was inhibited by PolRI and also SamA'B. CONCLUSION: A new system for SOS-independent overexpression of the activated DNA polymerases RI and SamA'B and their chimeras in the hisG46 Ames test background has been established and validated with several representative mutagens. Overall, the TA100 strain showed the highest sensitivity towards most tested mutagens. The observed inhibition of azide mutagenesis by PolRI* suggests that this type of Y-family DNA polymerases can perform also "corrective" error free replication on a damaged DNA.

Opposing roles of Y-family DNA polymerases in lipid peroxide mutagenesis at the hisG46 target in the Ames test.

DNA polymerases play a key role in mutagenesis by performing translesion DNA synthesis (TLS). The Y-family of DNA polymerases comprises several evolutionarily conserved families, specializing in TLS of different DNA adducts. Exocyclic etheno and propano DNA adducts are among the most common endogenous DNA lesions induced by lipid peroxidation reactions triggered by oxidative stress. We have investigated the participation of two enterobacterial representatives of the PolIV and PolV branches of Y-family DNA polymerases in mutagenesis by two model lipid peroxidation derived genotoxins, glyoxal and crotonaldehyde. Mutagenesis by the ethano adduct (glyoxal-derived) and the propano adduct (crontonaldehyde-derived) at the GC target in the Ames test depended exclusively on PolV type DNA polymerases such as PolRI. In contrast, PolIV suppressed glyoxal and, even more, crotonaldehyde mutagenesis, as detected by enzyme overexpression and gene knockout approaches. We propose that DNA polymerase IV, which is the mammalian DNA polymerase κ ortholog, acts as a housekeeper protecting the genome from lipoxidative stress.

Mutagenicity of ω-3 fatty acid peroxidation products in the Ames test.

Polyunsaturated fatty acids (PUFA) represent one of the main building blocks of cellular membranes and their varying composition impacts lifespan as well as susceptibility to cancer and other degenerative diseases. Increased intake of ω-3 PUFA is taught to compensate for the abundance of ω-6 PUFA in modern human diet and prevent cardiocirculatory diseases. However, highly unsaturated PUFA of marine and seed origin easily oxidize to aldehydic products which form DNA adducts. With increased PUFA consumption it is prudent to re-evaluate ω-3 PUFA safety and the genotoxic hazards of their metabolites. We have used the standard Ames test to examine the mutagenicity of 2 hexenals derived from lipid peroxidation of the common ω-3 PUFA in human diet and tissues. Both 4-hydroxyhexenal and 2-hexenal derived from the ω-3 docosahexaenoic and α-linolenic acid, respectively, induced base substitutions in the TA104 and TA100 Ames strains in a dose dependent manner. Their mutagenicity was dependent on the Y-family DNA polymerase RI and they did not induce other types of mutations such as the -2 and -1 frameshifts in the TA98 and TA97 strains. Our results expand previous findings about the mutagenicity of related ω-3 peroxidation product 4-oxohexenal and raise alert that overuse of ω-3 rich oils may have adverse effect on genome stability.

Epigenetic mutagen as histone modulator can be detected by yeast flocculation.

We have previously reported that flocculation of a yeast co-transformed with the human DNA methyltransferase 1 (DNMT1) and DNMT3B genes was inhibited by DNMT inhibitors. It is well known that epigenetic mutagens can disturb nucleosome positioning via DNA methylation and/or histone modification. In this study we first examined the effects of trichostatin A (TSA), a histone deacetylase inhibitor, on the flocculation level of yeast. TSA dose-dependently promoted the flocculation exhibited by the yeast transformed with the DNMT genes or empty vectors. Furthermore, TSA induced the expression of the flocculin-encoding gene FLO1 The anthracene-derived alizarin, a natural madder root dye, has a potential for carcinogenesis promotion; however, the mode of action has not been elucidated. It is considered that epigenetic changes can promote cancer. Alizarin but not anthracene enhanced the flocculation level of the yeast. Similar to TSA, alizarin also upregulated FLO1 mRNA. Surprisingly, western blotting indicated that alizarin, but not anthracene, reduced the level of histone H3 in yeast, and alizarin-treated cells frequently displayed abnormally shaped nuclei. These findings suggest that alizarin uniquely influences nucleosome structure. Taken together with our previous findings, this study suggests that the DNMT gene-transformed yeast strains are a useful tool for screening various classes of epigenetic mutagens.

Exclusive induction of G:C to A:T transitions by 3-azido-1,2-propanediol in yeast.

Sodium azide is a strong mutagen which has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolized to azidoalanine, but further bioactivation to a putative ultimate mutagen as well as the nature of the induced DNA modifications leading to mutations remain elusive. In this study, mutations induced in the CAN1 gene of yeast Saccharomyces cerevisiae by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) have been sequenced. Analysis of the forward mutation spectrum to canavanine resistance revealed that AZG induced nearly exclusively G:C to A:T transitions. AZG also induced reversions to tryptophan prototrophy by base-pair substitutions in a dose-dependent manner. This unusual mutational specificity may be shared by other organic azido compounds.

Escherichia coli DNA polymerase III is responsible for the high level of spontaneous mutations in mutT strains.

Reactive oxygen species induce oxidative damage in DNA precursors, i.e. dNTPs, leading to point mutations upon incorporation. Escherichia coli mutT strains, deficient in the activity hydrolysing 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP), display more than a 100-fold higher spontaneous mutation frequency over the wild-type strain. 8-oxo-dGTP induces A to C transversions when misincorporated opposite template A. Here, we report that DNA pol III incorporates 8-oxo-dGTP ≈ 20 times more efficiently opposite template A compared with template C. Single, double or triple deletions of pol I, pol II, pol IV or pol V had modest effects on the mutT mutator phenotype. Only the deletion of all four polymerases led to a 70% reduction of the mutator phenotype. While pol III may account for nearly all 8-oxo-dGTP incorporation opposite template A, it only extends ≈ 30% of them, the remaining 70% being extended by the combined action of pol I, pol II, pol IV or pol V. The unique property of pol III, a C-family DNA polymerase present only in eubacteria, to preferentially incorporate 8-oxo-dGTP opposite template A during replication might explain the high spontaneous mutation frequency in E. coli mutT compared with the mammalian counterparts lacking the 8-oxo-dGTP hydrolysing activities.

Origins of age-related DNA damage and dietary strategies for its reduction.

The polyunsaturated fatty acids in biological membranes serve as both the target and source of oxidative damage and can be regarded as the most unstable class of biomolecules in the body. Lipid peroxides arising from both spontaneous and enzymatic oxidation of polyunsaturated fatty acids are the major source of endogenous DNA damage linked to various age-related pathologies and initiating carcinogenesis. Here we describe the major types of lipid peroxide-derived DNA adducts and propose a simple dietary strategy to reduce their formation. This may be particularly beneficial to the aging organism, which has progressively impaired natural protective systems.

Efficient and erroneous incorporation of oxidized DNA precursors by human DNA polymerase eta.

Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis.

Involvement of Y-family DNA polymerases in mutagenesis caused by oxidized nucleotides in Escherichia coli.

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Visualization of the interaction between archaeal DNA polymerase and uracil-containing DNA by atomic force microscopy.

Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.

Processing of DNA lesions by archaeal DNA polymerases from Sulfolobus solfataricus.

Spontaneous damage to DNA as a result of deamination, oxidation and depurination is greatly accelerated at high temperatures. Hyperthermophilic microorganisms constantly exposed to temperatures exceeding 80 degrees C are endowed with powerful DNA repair mechanisms to maintain genome stability. Of particular interest is the processing of DNA lesions during replication, which can result in fixed mutations. The hyperthermophilic crenarchaeon Sulfolobus solfataricus has two functional DNA polymerases, PolB1 and PolY1. We have found that the replicative DNA polymerase PolB1 specifically recognizes the presence of the deaminated bases hypoxanthine and uracil in the template by stalling DNA polymerization 3-4 bases upstream of these lesions and strongly associates with oligonucleotides containing them. PolB1 also stops at 8-oxoguanine and is unable to bypass an abasic site in the template. PolY1 belongs to the family of lesion bypass DNA polymerases and readily bypasses hypoxanthine, uracil and 8-oxoguanine, but not an abasic site, in the template. The specific recognition of deaminated bases by PolB1 may represent an initial step in their repair while PolY1 may be involved in damage tolerance at the replication fork. Additionally, we reveal that the deaminated bases can be introduced into DNA enzymatically, since both PolB1 and PolY1 are able to incorporate the aberrant DNA precursors dUTP and dITP.

Erroneous incorporation of oxidized DNA precursors by Y-family DNA polymerases.

Deranged oxidative metabolism is a property of many tumour cells. Oxidation of the deoxynucleotide triphosphate (dNTP) pool, as well as DNA, is a major cause of genome instability. Here, we report that two Y-family DNA polymerases of the archaeon Sulfolobus solfataricus strains P1 and P2 incorporate oxidized dNTPs into nascent DNA in an erroneous manner: the polymerases exclusively incorporate 8-OH-dGTP opposite adenine in the template, and incorporate 2-OH-dATP opposite guanine more efficiently than opposite thymine. The rate of extension of the nascent DNA chain following on from these incorporated analogues is only slightly reduced. These DNA polymerases have been shown to bypass a variety of DNA lesions. Thus, our results suggest that the Y-family DNA polymerases promote mutagenesis through the erroneous incorporation of oxidized dNTPs during DNA synthesis, in addition to facilitating translesion DNA synthesis. We also report that human DNA polymerase eta, a human Y-family DNA polymerase, incorporates the oxidized dNTPs in a similar erroneous manner.