Mutagenesis and deoxyribonucleotide pool imbalance

Numerous studies have demonstrated that DNA-precursor pool imbalances are mutagenic and can modulate the lethality and mutagenicity of DNA-damaging agents. In addition, physical and chemical mutagens can induce alterations in DNA-precursor levels. Such findings suggest that regulation of intracellular concentrations of DNA precursors may be an important factor in environmental mutagenesis. In this article, results linking mutation and disturbances in DNA-precursor pools are reviewed.     

DNA single-strand breaks,double-strand breaks,and crosslinks in rat testicular germ cells: Measurements of their formation and repair by alkaline and neutral filter elution

This work describes a neutral and alkaline elution method for measuring DNA single-strand breaks (SSBs), DNA double-strand breaks (DSBs), and DNA-DNA crosslinks in rat testicular germ cells after treatments in vivo or in vitro with both chemical mutagens and gamma-irradiation. The methods depend upon the isolation of testicular germ cells by collagenase and trypsin digestion, followed by filtration and centrifugation. ¹³⁷Cs irradiation induced both DNA SSBs and DSBs in germ cells held on ice in vitro. Irradiation of the whole animal indicated that both types of DNA breaks are induced in vivo and can be repaired. A number of germ cell mutagens induced either DNA SSBs, DSBs, or cross-links after in vivo and in vitro dosing. These chemicals included methyl methane sulfonate, ethyl methane sulfonate, ethyl nitrosurea, dibromochlorpropane, ethylene dibromide, triethylene melamine, and mitomycin C. These results suggest that the blood-testes barrier is relatively ineffective for these mutagens, which may explain in part their in vivo mutagenic potency.This assay should be a useful screen for detecting chemical attack upon male germ-cell DNA and thus, it should help in the assessment of the mutagenic risk of chemicals. In addition, this approach can be used to study the processes of SSB, DSB, and crosslink repair in DNA of male germ cells, either from all stages or specific stages of development.Abbreviations DBCP dibromochlorpropane - DSB(s) DNA double-strand break(s) - EDB ethylene dibromide - EMS ethyl methane sulfonate - ENU ethyl nitrosurea - MC mitomycin C - MMS methyl methane sulfonate - SDS sodium dodecyl sulfate - SSB (s) DNA single-strand break(s) - TEM triethylene melamine - UDS unscheduled DNA synthesis     

Non-mutagenic nitroheterocycles. The lack of correlation between bacterial mutagenicity and one-electron reduction potential

The bacterial mutagenicity (Ames test) and the one-electron reduction potential for several nitropyridines, nitropyrazines and nitropyrimidines were determined. It was found that, although these nitroheterocycles underwent one-electron reduction more easily than metronidazole, they were not mutagenic in the Ames test. Six-membered ring nitroheterocycles, will, in general, exhibit diminished mutagenic potential compared to five-membered ring nitroheterocycles, and it is postulated that this effect is due to differences in the electronic nature and potential chemical reactivity of their respective nitrenium ions.     

Formaldehyde-induced mutagenesis: a novel mechanism for its action

A novel and unique mechanism for formaldehyde-induced mutagenesis is described which is mediated by the formation of an N6-substituted adenine ribonucleoside analogue, N6-hydroxymethyl adenosine, after an in vitro reaction of formaldehyde with adenosine. This type of ribonucleoside analogue (the deoxyribose derivative is ineffective) exhibits a powerful and remarkable germ-cell-stage-specific mutagenic effect in male Drosophila larvae, apparently by interfering with DNA repair. Circumstantial evidence is presented which indicates that the analogue most probably acts by its utilisation in the synthesis of diadenosine tetraphosphate (Ap4A) to form an antimetabolite(s) of Ap4A which subsequently interferes with Ap4A-mediated intracellular events, amongst which an effect on DNA repair would appear to be its mutagenic mechanism of action.     

Azidoalanine mutagenicity in Salmonella: Effect of homologation and α-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of l-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free l-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the α-methyl analogue, α-methylazidialanine, and the homologue, 2-amino-4-azidobutonoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that α-methyl substitution effectively blocks the mutagenic effects of azidoalanine with α-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the l-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.     

Azidoalanine mutagenicity in Salmonella: effect of homologation and alpha-methyl substitution

Azide mutagenicity in susceptible non-mammalian systems involves the requisite formation of L-azidoalanine, a novel mutagenic amino acid. The biochemical mechanism(s) of azidoalanine-induced mutagenesis, however, is not known. Previous studies of the structural requirements for azidoalanine mutagenicity suggested the importance of free L-amino acid character, and that bioactivation of azidoalanine to the ultimate mutagenic species is required. To gain more insight into possible enzymatic processing, the alpha-methyl analogue, alpha-methyl-azidoalanine, and the homologue, 2-amino-4-azidobutanoic acid, were synthesized and tested for mutagenic potency in Salmonella typhimurium strain TA1530. In addition, azidoacetic acid, a possible azidoalanine metabolite, was prepared and tested. The results show that alpha-methyl substitution effectively blocks the mutagenic effects of azidoalanine with alpha-methyl-azidoalanine being nearly devoid of mutagenic activity. In contrast, homologation of azidoalanine to yield 2-amino-4-azidobutanoic acid produces a marked increase in molar mutagenic potency. As with azidoalanine, the mutagenic activity of this homologue is associated with the L-isomer. Azidoacetic acid, however, was only very weakly mutagenic when tested as either the free acid or ethyl ester. This low mutagenic potency may indicate that bioactivation does not involve the entry of azide-containing azidoalanine catabolite into the Kreb's cycle. The high potency of 2-amino-4-azidobutanoic acid may be indicative of more efficient bioactivation and/or greater intrinsic activity. Importantly, the latter finding clearly shows that potent azido-amino acid mutagenicity is not limited to azidoalanine alone.     

Robustness and modularity properties of a non-covalent DNA catalytic reaction

The biophysics of nucleic acid hybridization and strand displacement have been used for the rational design of a number of nanoscale structures and functions. Recently, molecular amplification methods have been developed in the form of non-covalent DNA catalytic reactions, in which single-stranded DNA (ssDNA) molecules catalyze the release of ssDNA product molecules from multi-stranded complexes. Here, we characterize the robustness and specificity of one such strand displacement-based catalytic reaction. We show that the designed reaction is simultaneously sensitive to sequence mutations in the catalyst and robust to a variety of impurities and molecular noise. These properties facilitate the incorporation of strand displacement-based DNA components in synthetic chemical and biological reaction networks.     

Telomeric repeat mutagenicity in human somatic cells is modulated by repeat orientation and G-quadruplex stability

Telomeres consisting of tandem guanine-rich repeats can form secondary DNA structures called G-quadruplexes that represent potential targets for DNA repair enzymes. While G-quadruplexes interfere with DNA synthesis in vitro, the impact of G-quadruplex formation on telomeric repeat replication in human cells is not clear. We investigated the mutagenicity of telomeric repeats as a function of G-quadruplex folding opportunity and thermal stability using a shuttle vector mutagenesis assay. Since single-stranded DNA during lagging strand replication increases the opportunity for G-quadruplex folding, we tested vectors with G-rich sequences on the lagging versus the leading strand. Contrary to our prediction, vectors containing human [TTAGGG]₁₀ repeats with a G-rich lagging strand were significantly less mutagenic than vectors with a G-rich leading strand, after replication in normal human cells. We show by UV melting experiments that G-quadruplexes from ciliates [TTGGGG]₄ and [TTTTGGGG]₄ are thermally more stable compared to human [TTAGGG]₄. Consistent with this, replication of vectors with ciliate [TTGGGG]₁₀ repeats yielded a 3-fold higher mutant rate compared to the human [TTAGGG]₁₀ vectors. Furthermore, we observed significantly more mutagenic events in the ciliate repeats compared to the human repeats. Our data demonstrate that increased G-quadruplex opportunity (repeat orientation) in human telomeric repeats decreased mutagenicity, while increased thermal stability of telomeric G-quadruplexes was associated with increased mutagenicity.     

Similar mutagenicity of photoactivated porphyrins and ultraviolet A radiation in mouse embryonic fibroblasts: involvement of oxidative DNA lesions in mutagenesis

Ultraviolet A (UVA) radiation is implicated in the etiology of human skin cancer. However, the underlying mechanism of carcinogenicity for UVA is not fully delineated. A mutagenic role for UVA has been suggested, which involves activation of endogenous photosensitizers generating oxidative DNA damage. We investigated the mutagenicity of UVA alone and in combination with delta-aminolevulinic acid (delta-ALA), a precursor of the intracellular photosensitizers porphyrins, in transgenic Big Blue mouse embryonic fibroblasts. A significant generation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a typical promutagenic oxidative DNA lesion, was observed in cells treated with a combination of delta-ALA (1 mM) and UVA (0.06 J/cm(2)) as quantified by high-pressure liquid chromatography-tandem mass spectrometry (p < 0.001; relative to the control). The steady-state level of 8-oxo-dG, however, remained unchanged in cells irradiated with UVA or treated with delta-ALA alone. Other photolesions including cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts were not detectable in cells treated with delta-ALA and/or irradiated with UVA as determined by terminal transferase-dependent polymerase chain reaction assay. Mutation analyses of the cII transgene in cells treated with a combination of delta-ALA and UVA showed an approximately 3-fold increase in mutant frequency relative to the control (p < 0.008), as well as a unique induced mutation spectrum as established by DNA sequence analysis (p < 0.005; 95% CI, 0.002-0.009). No mutagenic effects were observed in cells irradiated with UVA or treated with delta-ALA alone. The spectrum of mutations produced by delta-ALA plus UVA was characterized by a significantly increased frequency of G --> T transversions (p < 0.0003; relative to the control), which are the hallmark mutations induced by 8-oxo-dG. Notably, the 8-oxo-dG-mediated mutagenicity of UVA plus delta-ALA is similar to that established previously for UVA alone at a mutagenic dose of 18 J/cm(2). We conclude that, in the presence of exogenous photosensitizers, UVA at a nonmutagenic dose induces mutations through the same mechanism as does a mutagenic dose of UVA per se.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

Kinetics of heterochiral strand displacement from PNA–DNA heteroduplexes

Dynamic DNA nanodevices represent powerful tools for the interrogation and manipulation of biological systems. Yet, implementation remains challenging due to nuclease degradation and other cellular factors. Use of l-DNA, the nuclease resistant enantiomer of native d-DNA, provides a promising solution. On this basis, we recently developed a strand displacement methodology, referred to as ‘heterochiral’ strand displacement, that enables robust l-DNA nanodevices to be sequence-specifically interfaced with endogenous d-nucleic acids. However, the underlying reaction – strand displacement from PNA–DNA heteroduplexes – remains poorly characterized, limiting design capabilities. Herein, we characterize the kinetics of strand displacement from PNA–DNA heteroduplexes and show that reaction rates can be predictably tuned based on several common design parameters, including toehold length and mismatches. Moreover, we investigate the impact of nucleic acid stereochemistry on reaction kinetics and thermodynamics, revealing important insights into the biophysical mechanisms of heterochiral strand displacement. Importantly, we show that strand displacement from PNA–DNA heteroduplexes is compatible with RNA inputs, the most common nucleic acid target for intracellular applications. Overall, this work greatly improves the understanding of heterochiral strand displacement reactions and will be useful in the rational design and optimization of l-DNA nanodevices that operate at the interface with biology.     

Unwinding of unnatural substrates by a DNA helicase

Helicases separate double-stranded DNA into single-stranded DNA intermediates that are required during replication and recombination. These enzymes are believed to transduce free energy available from ATPase activity to unwind the duplex and translocate along the nucleic acid lattice. The nature of enzyme-substrate interactions between helicases and duplex DNA substrates has not been well-defined. Most helicases require a single-stranded DNA overhang adjacent to duplex DNA in order to initiate unwinding. The strand containing the overhang is referred to as the loading strand whereas the complementary strand is referred to as the displaced strand. We have investigated the interactions between a DNA helicase and the DNA substrate by replacing the displaced strand with a nucleic acid mimic, peptide nucleic acid (PNA). PNA is capable of forming duplex structures with DNA according to Watson-Crick base pairing rules, but contains a N-(2-aminoethyl)glycine backbone in place of the deoxyribose phosphates. The PNA-DNA hybrids had higher melting temperatures than their DNA-DNA counterparts. Dda helicase, from bacteriophage T4, was able to unwind the DNA-PNA substrates at similar rates as DNA-DNA substrates. The results indicate that the rate-limiting step for unwinding is relatively insensitive to the chemical nature of the displaced strand and the thermal stability of oligonucleotide substrates.     

Mutagenicity of irradiated solutions of nucleic acid bases and nucleosides in Salmonella typhimurium

Solutions of nucleic acid bases, nucleosides and a nucleotide, saturated with either N2, N2O or O2, were irradiated and tested for mutagenicity towards Salmonella typhimurium, with and without pre-incubation. Irradiated solutions of the nucleic acid bases were all non-mutagenic. Irradiated solutions of the nucleosides showed mutagenicity in S. typhimurium TA100 (pre-incubation assay). Generally, the mutagenicity followed the order: N2O greater than N2 greater than O2. The results show that the formation of mutagenic radiolytic products is initiated by attack of mainly OH radicals on the 2-deoxy-D-ribose moiety of the nucleosides. With irradiated solutions of the nucleotide, thymidine-5'-monophosphate, no mutagenicity could be detected.     

Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP.

An analysis is presented in which are evaluated correlations among chemical structure, mutagenicity to Salmonella, and carcinogenicity to rats and mice among 301 chemicals tested by the U.S. NTP. Overall, there was a high correlation between structural alerts to DNA reactivity and mutagenicity, but the correlation of either property with carcinogenicity was low. If rodent carcinogenicity is regarded as a singular property of chemicals, then neither structural alerts nor mutagenicity to Salmonella are effective in its prediction. Given this, the database was fragmented and new correlations sought between the derived sub-groups. First, the 301 chemicals were segregated into six broad chemical groupings. Second, the rodent cancer data were partially segregated by target tissue. Using the previously assigned structural alerts to DNA reactivity (electrophilicity), the chemicals were split into 154 alerting chemicals and 147 non-alerting chemicals. The alerting chemicals were split into three chemical groups; aromatic amino/nitro-types, alkylating agents and miscellaneous structurally-alerting groups. The non-alerting chemicals were subjectively split into three broad categories; non-alerting, non-alerting containing a non-reactive halogen group, and non-alerting chemical with minor concerns about a possible structural alert. The tumor data for all 301 chemicals are re-presented according to these six chemical groupings. The most significant findings to emerge from comparisons among these six groups of chemicals were as follows: (a) Most of the rodent carcinogens, including most of the 2-species and/or multiple site carcinogens, were among the structurally alerting chemicals. (b) Most of the structurally alerting chemicals were mutagenic; 84% of the carcinogens and 66% of the non-carcinogens. 100% of the 33 aromatic amino/nitro-type 2-species carcinogens were mutagenic. Thus, for structurally alerting chemicals, the Salmonella assay showed high sensitivity and low specificity (0.84 and 0.33, respectively). (c) Among the 147 non-alerting chemicals less than 5% were mutagenic, whether they were carcinogens or non-carcinogens (sensitivity 0.04).     

The strand transfer oligonucleotide inhibitors of HIV-integrase

Retroviral integrase participates in two catalytic reactions, which require interactions with the two ends of the viral DNA in the 3′processing reaction, and with a targeted host DNA in the strand transfer reaction. The 3′-hydroxyl group of 2′-deoxyadenosine resulting from the specific removing of GT dinucleotide from the viral DNA in the processing reaction provides the attachment site for the host DNA in a transesterification reaction. We synthesized oligonucleotides (ONs) of various lengths that mimic the processed HIV-1 U5 terminus of the proviral long terminal repeat (LTR) and are ended by 2′-deoxyadenosine containing a 3′-O-phosphonomethyl group. The duplex stability of phosphonomethyl ONs was increased by covalent linkage of the modified strand with its complementary strand by a triethylene glycol loop (TEG). Modified ONs containing up to 10 bases inhibited in vitro the strand transfer reaction catalyzed by HIV-1 integrase at nanomolar concentrations.     

Sulfur-containing proreactive intermediates: hydrolysis and mutagenicity of halovinyl 2-nitrophenyl disulfides

Chemical cleavage of the sulfur-sulfur bond in halovinyl and fluoroalkyl 2-nitrophenyl disulfides is expected to yield halovinyl and fluoroalkyl thiols identical to those formed by cysteine conjugate beta-lyase catalyzed cleavage of the corresponding cysteine S-conjugates. To study the potential use of disulfides as precursors for these thiols, whose transformation to acylating agents is most likely responsible for cysteine S-conjugate mutagenicity, we determined the mutagenicity of several halovinyl and fluoroalkyl 2-nitrophenyl disulfides and identified products formed by hydrolysis of these disulfides, 1,2,3,4,4-Pentachlorobutadienyl 2-nitrophenyl disulfide, 1,2,2-trichlorovinyl 2-nitrophenyl disulfide, 1-fluro-2,2-dichlorovinyl 2-nitrophenyl disulfide and 1,2-dichloro-3,3,3-trifluropropenyl 2-nitrophenyl disulfide were mutagenic in nitroreductase deficient strains of Salmonella typhimurium TA100; as haloalkyl cysteine S-conjugates, 1,1-difluoro-2,2-dichloroethyl 2-nitrophenyl disulfide and 1-chloro-1,2,2-trifluroethyl 2-nitrophenyl disulfide were not mutagenic. Hydrolysis of 1,2,3,4,4-pentachlorobutadienyl 2-nitrophenyl disulfide and 1,2,2-trifluorethyl 2-nitrophenyl disulfide in presence of diethylamine resulted in tetrachlorothiobutenoic acid diethylamide and chlorofluorothionoacetic acid diethylamide. The differences in mutagenicity between halovinyl and fluoroalkyl disulfides are most likely responsible to their different abilities to react with DNA-constituents. Products formed from the mutagenic 1,2,3,4,4-pentachlorobutadienyl 2-nitrophenyl disulfide modified 2'-deoxyguanosine-3'-monophosphate and DNA as detected by 32Phosphorus-postlabeling, whereas products formed from the nonmutagenic 1-chloro-1,2,2-trifluoroethyl 2-nitrophenyl disulfide did not result in detectable 2'-deoxyguanosine-3'-monophosphate and DNA modification.     

Caged single and double strand breaks

Ionizing radiation and radiomimetic drugs such as bleomycin, calichieamycin, neocarzinostatin chromophore, and other synthetic agents can produce both single and double strand breaks in DNA. The ability to study the structure-activity relationships of single and double-strand break repair, lethality, and mutagenesis in vivo is complicated by the numerous types and sites of DNA cleavage products that can be induced by such agents. The ability to "cage" such breaks in DNA might help to further such studies and additionally afford a mechanism for activating and deactivating nucleic acid based drugs and probes. The major type of single strand break induced by ionizing radiation is a 3'- and 5'-phosphate terminated single nucleotide gap. Previously, a caged strand break of this type had been developed that was designed to produce the 5'-phosphate directly upon irradiation with 366 nm light, and the 3'-phosphate by a subsequent beta-elimination reaction [Ordoukhanian, P., and Taylor, J.-S. (1995) J. Am. Chem. Soc. 117, 9570]. Unfortunately, the release of the 3'-phosphate group was quite slow at pH 7. To circumvent this problem, a second caged strand break has been developed that produces the 3'-phosphate directly upon irradiation, and the 5'-phosphate by a subsequent beta-elimination reaction. When this caged strand break was used in tandem with the previous caged strand break, 5'- and 3'-phosphate terminated gaps could be directly produced by irradiation with 366 nm light. These caged single strand breaks were also incorporated in tandem into hairpin substrates to demonstrate that they could be used to cage double strand breaks. These caged single strand breaks should be generally useful for generating site-specific DNA single and double strand breaks and gaps, using wavelengths and doses of light that are nondetrimental to biological systems. Because the position of the single strand break can be varied, it should now be possible to examine the effect of the sequence context and cleavage pattern of single and double strand breaks on the lethality and mutagenicity of this important class of DNA damage.     

Use of nitrous acid-dependent decrease in mutagenicity as an indication of the presence of mutagenic primary aromatic amines. Non-specific reactions with phenols and benzo[alpha]pyrene

Treatment of mutagenic primary aromatic amines with nitrous acid is known to decrease their mutagenicity. We examined some factors concerning the validity of using decreases in mutagenicity due to nitrous acid treatment as an indication of the presence of mutagenic primary aromatic amines in complex mixtures. We found that treatment of benzo[alpha]pyrene with nitrous acid for the extended periods of time previously employed leads to formation of three nitrobenzo[alpha]pyrene isomers. Some of the isomers are direct-acting mutagens for S. typhimurium with considerably greater mutagenicity than benzo[alpha]pyrene isomers. In attempts to minimize reaction of chemicals other than aromatic amines, we found that only very brief reaction periods are required for complete reaction of nitrous acid with representative aromatic amines, essentially eliminating their mutagenicity. During such brief reaction periods modification of benzo[alpha]pyrene is negligible, but phenols react readily. Chromatographic analysis indicated that reaction of nitrous acid with aromatic amines leads to the formation of families of products, thereby increasing the complexity of the mixtures in which the amines may occur. Thus, experiments examining the effects of nitrous acid on the mutagenic activity of complex mixtures must be carefully designed, and the results must be interpreted cautiously.