Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines.

Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2'-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8-aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.

Selective Inbreeding: Genetic Crosses Drive Apparent Adaptive Mutation in the Cairns-Foster System of Escherichia coli.

The Escherichia coli system of Cairns and Foster employs a lac frameshift mutation that reverts rarely (10-9/cell/division) during unrestricted growth. However, when 108 cells are plated on lactose medium, the nongrowing lawn produces ∼50 Lac+ revertant colonies that accumulate linearly with time over 5 days. Revertants carry very few associated mutations. This behavior has been attributed to an evolved mechanism ("adaptive mutation" or "stress-induced mutagenesis") that responds to starvation by preferentially creating mutations that improve growth. We describe an alternative model, "selective inbreeding," in which natural selection acts during intercellular transfer of the plasmid that carries the mutant lac allele and the dinB gene for an error-prone polymerase. Revertant genome sequences show that the plasmid is more intensely mutagenized than the chromosome. Revertants vary widely in their number of plasmid and chromosomal mutations. Plasmid mutations are distributed evenly, but chromosomal mutations are focused near the replication origin. Rare, heavily mutagenized, revertants have acquired a plasmid tra mutation that eliminates conjugation ability. These findings support the new model, in which revertants are initiated by rare pre-existing cells (105) with many copies of the F'lac plasmid. These cells divide under selection, producing daughters that mate. Recombination between donor and recipient plasmids initiates rolling-circle plasmid over-replication, causing a mutagenic elevation of DinB level. A lac+ reversion event starts chromosome replication and mutagenesis by accumulated DinB. After reversion, plasmid transfer moves the revertant lac+ allele into an unmutagenized cell, and away from associated mutations. Thus, natural selection explains why mutagenesis appears stress-induced and directed.

Half-Intercalation Stabilizes Slipped Mispairing and Explains Genome Vulnerability to Frameshift Mutagenesis by Endogenous "Molecular Bookmarks".

Some 60 years ago chemicals that intercalate between base pairs of duplex DNA were found to amplify frameshift mutagenesis. Surprisingly, the robust induction of frameshifts by intercalators still lacks a mechanistic model, leaving this classic phenomenon annoyingly intractable. A promising idea of asymmetric half-intercalation-stabilizing frameshift intermediates during DNA synthesis has never been developed into a model. Instead, researchers of frameshift mutagenesis embraced the powerful slipped-mispairing concept that unexpectedly struggled with the role of intercalators in frameshifting. It is proposed that the slipped mispairing and the half-intercalation ideas are two sides of the same coin. Further, existing findings are reviewed to test predictions of the combined "half-intercalation into the slipped-mispairing intermediate" model against accumulated knowledge. The existence of potential endogenous intercalators and the phenomenon of "DNA bookmarks" reveal ample possibilities for natural frameshift mutagenisis in the cell. From this alarming perspective, it is discussed how the cell could prevent genome deterioration from frameshift mutagenesis.

Assessment of antimutagenic action of Celtis glabrata Steven ex Planch. (Cannabaceae) extracts against base pair exchange and frame shift mutations on Salmonella typhimurium TA98 and TA100 strains by Ames test.

CONTEXT: Celtis glabrata is used in Turkey for the treatment of various health disorders. OBJECTIVE: The acetone, chloroform, ethanol, and methanol extracts of C. glabrata leaf, fruit, and seed were investigated to evaluate their antimutagenic activities. MATERIAL AND METHODS: The antimutagenicity of these extracts was determined by Ames test against mutagens (4-nitro-O-phenylenediamine, 2-aminofluorene (2-AF), and sodium azide (SA)). The extracts were used at concentrations between 5 and 0.005 mg/plate. RESULTS: The ethanol extracts of leaves exhibited strong antimutagenicity (70%) against 2-AF with S9 at 5 mg/plate on TA98. But methanol (61%, 53%) and acetone (53%, 52%) also revealed strong inhibition rates at concentrations of ≥ 0.5 mg/plate. Among the extracts, the highest activity (96%) was obtained from acetone extract against SA without S9, followed by chloroform extract (91%) at a dose of 5 mg/plate on TA100 with S9. Ethanol (without S9) and chloroform (with S9) extracts showed strong antimutagenicity at all doses. Exception of chloroform and acetone (without S9), all fruit extracts (with/without S9) manifested strong antimutagenicity at doses of ≥ 0.5 mg/plate on TA98 strain. Ethanol extracts revealed 68% inhibition against 2-AF on TA98. Acetone and ethanol extracts manifested 84% and 82% inhibition against SA on TA100, respectively. All the extracts of seeds revealed strong inhibition against 2-AF at ≥ 0.5 mg/plate doses on TA98, but acetone extract showed excellent antimutagenicity (94%). Moreover, the chloroform (74, 73, 63, 54%), acetone (74, 72, 70, 65%) and methanol (74, 67, 63, 61%) extracts of seeds revealed strong antimutagenic activity on TA100 against SA with S9. DISCUSSION AND CONCLUSION: This plant may be natural source of antimutagenic agents.

Dicholesteroyl diselenide: cytotoxicity, genotoxicity and mutagenicity in the yeast Saccharomyces cerevisiae and in Chinese hamster lung fibroblasts.

The organoselenium compound, dicholesteroyl diselenide (DCDS) is a structural analogue of diphenyl diselenide (DPDS) and may be considered as a promising antioxidant drug in vivo. Nevertheless, little is known about the toxicological properties of DCDS. In the present study we evaluated the cytotoxic, genotoxic and mutagenic properties of DCDS in Chinese hamster lung fibroblasts (V79) and in strains of the yeast Saccharomyces cerevisiae, proficient and deficient in several DNA-repair pathways. The results with V79 cells show that DCDS induced cytotoxicity, GSH depletion and elevation of lipid peroxidation at lower concentrations than did DPDS. DCDS also generated single- and double-strand DNA breaks in V79 cells, both in the presence and in the absence of metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, the induction of oxidative DNA base-damage was demonstrated by means of a modified comet assay with formamidopyrimidine-DNA glycosylase and endonuclease III. Treatment with DCDS also induced micronucleus formation in V79 cells as well as point and frame-shift mutations in a haploid wild-type strain of S. cerevisiae. Yeast mutants defective in base excision-repair proteins were the most sensitive to DCDS. Pre-incubation with N-acetylcysteine reduced DCDS's oxidative, genotoxic and mutagenic effects in yeast and in V79 cells. Our findings indicate that the presence of cholesteroyl substituents in DCDS results in elevation of its cytotoxic and genotoxic potential compared with that of DPDS in yeast and in V79 cells. However, due to dose-dependent contrasting behaviour of organoselenium compounds and differences in their toxicity in in vitro and in vivo systems, further studies are needed in order to establish the non-toxic concentration range for treatment in mammals.

The role of plant metabolism in the mutagenic and cytotoxic effects of four organophosphorus insecticides in Salmonella typhimurium and in human cell lines.

This study used a cell/microbe co-incubation assay to evaluate the effect of four organophosphorus insecticides (parathion-methyl, azinphos-methyl, omethoate, and methamidophos) metabolized by coriander (Coriandrum sativum). The reverse mutation of Salmonella typhimurium strains TA98 and TA100 was used as an indicator of genetic damage. Treatments with these insecticides inhibited peroxidase activity in plant cells by between 17% (omethoate) and 98% (azinphos-methyl) and decreased plant protein content by between 36% (omethoate) and 99.6% (azinphos-methyl). Azinphos-methyl was the most toxic when applied directly. In the Ames test, treatments applied directly to strain TA100 killed the bacteria; however, the presence of plant metabolism detoxified the system and permitted the growth of bacteria. In strain TA98, plant metabolites of insecticides were mutagenic. This result suggests that the tested pesticides produce mutations through frameshifting. The same pesticides were applied to human skin (HaCaT) and lung (NL-20) cell lines to evaluate their effects on cell viability. Pesticides applied directly were more cytotoxic than the combination of pesticide plus coriander metabolic fraction. Omethoate and methamidophos did not affect the viability of HaCaT cells, but azinphos-methyl and parathion-methyl at 100 and 1000µgmL(-1) significantly decreased viability (p<0.05). The NL-20 cell line was remarkably sensitive to the direct application of insecticides. All of the treatment conditions caused decreases in NL-20 cell viability (e.g., viability decreased to 12.0% after parathion-methyl treatment, to 14.7% after azinphos-methyl treatment, and to 6.9% after omethoate treatment). Similar to the Ames test, all of the insecticides showed decreased toxicity in human cells when they were cultured in the presence of plant metabolism. In conclusion, when the studied organophosphorus insecticides were plant-metabolized, they induced mutations in the bacterial strain TA98. In human cell lines, plant metabolism reduced the cytotoxic properties of the insecticides, and human keratinocytes were more resistant to mortality than bronchial cells.

Bone morphogenetic protein 3 controls insulin gene expression and is down-regulated in INS-1 cells inducibly expressing a hepatocyte nuclear factor 1A-maturity-onset diabetes of the young mutation.

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A-maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.

Anti- and pro-mutagenic effects of silymarin in the Ames bacterial reverse mutation assay.

Silymarin, a polyphenolic flavonoid isolated from milk thistle (Silybum marianum), is being used clinically in Europe and Asia for the treatment of liver diseases. Silymarin has a strong antioxidative action capable of scavenging both free radicals and reactive oxygen species responsible for cancer. Silymarin, a powerful hepatoprotective and antioxidant, was chosen in the present study and was tested for its antimutagenic activity using an in vitro test, the Ames bacterial reverse mutation assay. The results indicated that silymarin showed a significant mutagenicity in frame shift mutant strains (TA97a and TA98) with metabolic activation. This compound also showed stronger antimutagenic effect against 2-aminofluorene and 4-nitroquinoline N-oxide induced mutation. When pre-, co- and post-treatment of silymarin was carried out, it showed stronger antimutagenic activity in the post-treatment with 2-aminofluorene and 4-nitroquinoline N-oxide in TA97a and TA98 strains.

Interaction between the mismatch repair and nucleotide excision repair pathways in the prevention of 5-azacytidine-induced CG-to-GC mutations in Escherichia coli.

5-Azacytidine induces CG-to-GC transversion mutations in Escherichia coli. The results presented in this paper provide evidence that repair of the drug-induced lesions that produce these mutations involves components of both the mismatch repair and nucleotide excision repair systems. Strains deficient in mutL, mutS, uvrA, uvrB or uvrC all showed an increase in mutation in response to 5-azacytidine. Using a bacterial two-hybrid assay, we showed that UvrB interacts with MutL and MutS in a drug-dependent manner, while UvrC interacts with MutL independent of drug. We suggest that 5-azacytidine-induced mismatches recruit MutS and MutL, but are poorly processed by mismatch repair. Instead, the stalled MutS-MutL complex recruits the Uvr proteins to complete repair.

Transformation of MCF-10A cells by random mutagenesis with frameshift mutagen ICR191: a model for identifying candidate breast-tumor suppressors.

BACKGROUND: Widely accepted somatic mutation theory of carcinogenesis states that mutations in oncogenes and tumor suppressor genes in genomes of somatic cells is the cause of neoplastic transformation. Identifying frequent mutations in cancer cells suggests the involvement of mutant genes in carcinogenesis. RESULTS: To develop an in vitro model for the analysis of genetic alterations associated with breast carcinogenesis, we used random mutagenesis and selection of human non-tumorigenic immortalized breast epithelial cells MCF-10A in tissue-culture conditions that mimic tumor environment. Random mutations were generated in MCF-10A cells by cultivating them in a tissue-culture medium containing the frameshift-inducing agent ICR191. The first selective condition we used to transform MCF1-10A cells was cultivation in a medium containing mutagen at a concentration that allowed cell replication despite p53 protein accumulation induced by mutagen treatment. The second step of selection was either cell cultivation in a medium with reduced growth-factor supply or in a medium that mimics a hypoxia condition or growing in soft agar. Using mutagenesis and selection, we have generated several independently derived cultures with various degrees of transformation. Gene Identification by Nonsense-mediated mRNA decay Inhibition (GINI) analysis has identified the ICR191-induced frameshift mutations in the TP53, smoothelin, Ras association (RalGDS/AF-6) domain family 6 (RASSF6) and other genes in the transformed MCF-10A cells. The TP53 gene mutations resulting in the loss of protein expression had been found in all independently transformed MCF-10A cultures, which form large progressively growing tumors with sustained angiogenesis in nude mice. CONCLUSION: Identifying genes containing bi-allelic ICR191-induced frameshift mutations in the transformed MCF-10A cells generated by random mutagenesis and selection indicates putative breast-tumor suppressors. This can provide a model for studying the role of mutant genes in breast carcinogenesis.

Toxicity and mutagenicity of selenium compounds in Saccharomyces cerevisiae.

Selenium (Se) is an essential trace element for humans, animals and some bacteria which is important for many cellular processes. Se's bio-activity is mainly influenced by its chemical form and dose. The use of Se supplements in the human diet emphasizes the need to establish both the beneficial and detrimental doses of each Se compound. We have evaluated three different Se compounds, sodium selenite (SeL), selenomethionine (SeM) and Se-methylselenocysteine (SeMC), with respect to their potential DNA damaging effects. The budding yeast Saccharomyces cerevisiae was used as a model system to test the toxic and mutagenic effects as well as the DNA double-strand breakage potency of these Se compounds in both exponentially growing and stationary yeast cells. Only SeL manifested any significant toxic effects in the yeast which were more pronounced in the exponentially growing cells than in those cells in the stationary phase of growth. The toxic effects of SeL were however accompanied with the pro-mutagenic effects in the stationary cell phase of growth. The toxic and mutagenic effects of SeL are likely associated with the ability of this compound to generate DNA double-strand breaks (DSB). We also show that SeL significantly increased frame-shift mutations, especially 1-4 bp deletions, in the CAN1 mutational spectrum of the yeast genome when compared to untreated control. We propose that SeL is acting as an oxidizing agent in S. cerevisiae producing superoxide and oxidative damage to DNA accounting for the observed DSB and cell death.

Frameshift mutations produced by 9-aminoacridine in wild-type, uvrA and recA strains of Escherichia coli; specificity within a hotspot.

The endogenous tonB gene of Escherichia coli was used as a target for 9-aminoacridine-induced mutations that were identified in recA(-) and uvrA(-) cells. The cytotoxicity of 9-aminoacridine was enhanced in the uvrA and recA strains compared to the wild-type strain, and the mutagenicity of 9-aminoacridine in the uvrA and recA strains was similar to that in the wild type. For all three strains, the most common mutations were minus frameshifts in repetitive G:C base-pairs followed by minus frameshifts in nonrepetitive G:C base-pairs. 9-aminoacridine-induced minus frameshifts in the wild-type strain were distributed with several hot and warm spots. These sites were also hot and warm spots for minus frameshifts in the recA and uvrA stains. Furthermore, they were hot and warm sites in a 9-aminoacridine-treated strain carrying the target tonB gene oriented in the opposite direction. 9-Aminoacridine is known to interact with DNA to form intercalations which are involved in minus frameshift mutagenesis. In this study, we therefore argue that 1) 9-aminoacridine can induce bulky DNA lesions which are excised by nucleotide excision repair and not involved in mutagenesis, 2) the presence or absence of a recA-dependent repair pathway does not influence the mutagenic effect of 9-aminoacridine, and 3) both leading strand and lagging strand replication equally produce minus frameshifts, therefore gene orientation is not an important determinant of the formation of hot and warm spots by 9-aminoacridine.

Overexpression of DNA polymerase beta results in an increased rate of frameshift mutations during base excision repair.

DNA polymerase beta (Pol beta) is important for the base excision repair (BER) pathway. Overexpression of Pol beta is frequently found in cancer cells and is thought to be associated with tumorigenesis. In this study, we examined BER fidelity in extracts derived from a human lymphoblastoid cell line that over expresses Pol beta compared to normal control cells. Using an in vitro mutagenesis assay, we found an increased rate of frameshift mutations arising during DNA repair in whole-cell extracts derived from the Pol beta-overexpressing cells. We demonstrate that the addition of excess Pol beta to a control cell extract enhances the mutagenic potential of the extract. Furthermore, using cell extracts and purified Pol beta, we demonstrate that the mechanism of frameshift formation involves slippage of Pol beta during the one-nucleotide gap-filling step of BER and that this slippage is fixed by strand-displacement synthesis stimulated by an excess of Pol beta.

Antisense-induced ribosomal frameshifting.

Programmed ribosomal frameshifting provides a mechanism to decode information located in two overlapping reading frames by diverting a proportion of translating ribosomes into a second open reading frame (ORF). The result is the production of two proteins: the product of standard translation from ORF1 and an ORF1-ORF2 fusion protein. Such programmed frameshifting is commonly utilized as a gene expression mechanism in viruses that infect eukaryotic cells and in a subset of cellular genes. RNA secondary structures, consisting of pseudoknots or stem-loops, located downstream of the shift site often act as cis-stimulators of frameshifting. Here, we demonstrate for the first time that antisense oligonucleotides can functionally mimic these RNA structures to induce +1 ribosomal frameshifting when annealed downstream of the frameshift site, UCC UGA. Antisense-induced shifting of the ribosome into the +1 reading frame is highly efficient in both rabbit reticulocyte lysate translation reactions and in cultured mammalian cells. The efficiency of antisense-induced frameshifting at this site is responsive to the sequence context 5' of the shift site and to polyamine levels.

Mutator specificity of Escherichia coli alkB117 allele.

The Escherichia coli AlkB protein encoded by alkB gene was recently found to repair cytotoxic DNA lesions 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) by using a novel iron-catalysed oxidative demethylation mechanism that protects the cell from the toxic effects of methylating agents. Mutation in alkB results in increased sensitivity to MMS and elevated level of MMS-induced mutations. The aim of this study was to analyse the mutational specificity of alkB117 in a system developed by J.H. Miller involving two sets of E. coli lacZ mutants, CC101-106 allowing the identification of base pair substitutions, and CC107-CC111 indicating frameshift mutations. Of the six possible base substitutions, the presence of alkB117 allele led to an increased level of GC-->AT transitions and GC-->TA and AT-->TA transversions. After MMS treatment the level of GC-->AT transitions increased the most, 22-fold. Among frameshift mutations, the most numerous were -2CG, -1G, and -1A deletions and +1G insertion. MMS treatment appreciably increased all of the above types of frameshifts, with additional appearance of the +1A insertion.

Isolation and characterization of dominant and recessive IL-3-independent hematopoietic transformants.

Retroviral integration mutagenesis and treatment with the frameshift mutagen ICR191 were used to transform v-H-ras expressing PB-3c cells to interleukin-3 (IL-3) independence. Six clones displayed viral integrations into the 3' region of the IL-3 gene thus acting post-transcriptionally by disrupting the AU-rich instability element. Two clones contained reverse orientation integration into the raf-1 gene revealing an enhancer insertion mechanism. Growth by this mechanism was sensitive to the Raf-1 inhibitor BAY 43-9006 and the Mek inhibitor U0126. Following treatment with ICR191, IL-3-independent clones were recovered and studied by cell fusion. With 21/22 clones, IL-3 independence resulted from a recessive mechanism as cellular hybrids with parental cells reverted to IL-3 dependence. Recessive clone D2c displayed increased phospho-Erk1/2 levels and was growth sensitive to U0126, but not to BAY43-9006. The single dominant clone, D5a, showed no signs of mitogen-activated protein kinases pathway activation but displayed constitutive phosphorylation of Stat5. We conclude that PB-3c has several options to acquire IL-3 growth autonomy involving transcriptional or post-transcriptional mechanisms affecting the distal regulators Erk or Stat5. The reported panel of independent dominant and recessive transformants should provide a useful tool for inhibitor profiling.

Specificity of replicative and SOS-inducible DNA polymerases in frameshift mutagenesis: mutability of Salmonella typhimurium strains overexpressing SOS-inducible DNA polymerases to 30 chemical mutagens.

DNA replication is frequently hindered because of the presence of DNA lesions induced by endogenous and exogenous genotoxic agents. To circumvent the replication block, cells are endowed with multiple specialized DNA polymerases that can bypass a variety of DNA damage. To better understand the specificity of specialized DNA polymerases to bypass lesions, we have constructed a set of derivatives of Salmonella typhimurium TA1538 harboring plasmids carrying the polB, dinB or mucAB genes encoding Escherichia coli DNA polymerase II, DNA polymerase IV or DNA polymerase RI, respectively, and examined the mutability to 30 chemicals. The parent strain TA1538 possesses CGCGCGCG hotspot sequence for -2 frameshift. Interestingly, the chemicals could be classified into four groups based on the mutagenicity to the derivatives: group I whose mutagenicity was highest in strain YG5161 harboring plasmid carrying dinB; group II whose mutagenicity was almost equally high in strain YG5161 and strain TA98 harboring plasmid carrying mucAB; group III whose mutagenicity was highest in strain TA98; group IV whose mutagenicity was not affected by the introduction of any of the plasmids. Introduction of plasmid carrying polB did not enhance the mutagenicity except for benz[a]anthracene. We also introduced a plasmid carrying polA encoding E. coli DNA polymerase I to strain TA1538. Strikingly, the introduction of the plasmid reduced the mutagenicity of chemicals belonging to groups I, II and III, but not the chemicals of group IV, to the levels observed in the derivative whose SOS-inducible DNA polymerases were all deleted. These results suggest that (i) DNA polymerase IV and DNA polymerase RI possess distinct but partly overlapping specificity to bypass lesions leading to -2 frameshift, (ii) the replicative DNA polymerase, i.e., DNA polymerase III, participates in the mutagenesis and (iii) the enhanced expression of E. coli polA may suppress the access of Y-family DNA polymerases to the replication complex.

Sub-chronic exposure to 1,1-dichloropropene induces frameshift mutations in lambda transgenic medaka.

1,1-Dichloropropene (1,1-DCP) is a contaminant present in both ground and surface waters used as sources for drinking water. Structural similarity to several compounds with known mutagenicity and carcinogenicity, and recent demonstration of mutagenicity in vitro, suggest this compound may be similarly mutagenic in vivo. A transgenic fish model, the lamda transgenic medaka, was used to evaluate the potential mutagenicity of this contaminant in vivo following sub-chronic exposure for 6 weeks. Mutant frequencies of the cII target gene (MF) increased six-fold in the livers of fish exposed to the lowest 1,1-DCP exposure concentration (0.44 mg/L, MF = 18.4 x 10(-5), and increased with each treatment, culminating in a 32-fold induction in fish from the highest 1,1-DCP treatment (16.60 mg/L, MF = 96.3 x 10(-5). Mutations recovered from treated fish showed a distinctive mutational spectrum comprised predominantly of +1 frameshift mutations, induced 166-fold above that of untreated animals. The majority of frameshifts were +1 insertions at thiamine and adenine. These results represent the first evidence of mutagenicity of 1,1-DCP in vivo, and of the highly characteristic spectrum of induced mutations dominated by +1 frameshift mutations. Based upon results from previous in vitro studies, the similar role of glutathione S-transferase (GSTT1-1) in the activation of 1,1-DCP to a mutagen in vivo is also suggested. This study further illustrates the utility of the lamda transgenic medaka as a model for identifying and characterizing potential genetic health risks associated with chemical exposures in the environment.

Mechanism for N-acetyl-2-aminofluorene-induced frameshift mutagenesis by Escherichia coli DNA polymerase I (Klenow fragment).

N-Acetyl-2-aminofluorene (AAF) is a chemical carcinogen that reacts with guanines at the C8 position in DNA to form a structure that interferes with DNA replication. In bacteria, the NarI restriction enzyme recognition sequence (G1G2CG3CC) is a very strong mutational hot spot when an AAF adduct is positioned at G3 of this sequence, causing predominantly a -2 frameshift GC dinucleotide deletion mutation. In this study, templates were constructed that contained an AAF adduct at this position, and primers of different lengths were prepared such that the primer ended one nucleotide before or opposite or one nucleotide after the adduct site. Primer extension and gel shift binding assays were used to study the mechanism of bypass by the Escherichia coli DNA polymerase I (Klenow fragment) in the presence of these templates. Primer extension in the presence of all four dNTPs produced a fully extended product using the unmodified template, while with the AAF-modified template synthesis initially stalled at the adduct site and subsequent synthesis resulted in a product that contained the GC dinucleotide deletion. Extension product and gel shift binding analyses were consistent with the formation of a two-nucleotide bulge structure upstream of the active site of the polymerase after a nucleotide is incorporated across from the adduct. These data support a model in which the AAF adduct in the NarI sequence specifically induces a structure upstream of the polymerase active site that leads to the GC frameshift mutation and that it is this structure that allows synthesis past the adduct to occur.