supF Gene Research Articles

Targeted Gene Modification of Hematopoietic Progenitor Cells in Mice Following Systemic Administration of a PNA-peptide Conjugate

Hematopoietic stem cell (HSC) gene therapy offers promise for the development of new treatments for a variety of hematologic disorders. However, efficient in vivo modification of HSCs has proved challenging, thus imposing constraints on the therapeutic potential of this approach. Herein, we provide a gene-targeting strategy that allows site-specific in vivo gene modification in the HSCs of mice. Through conjugation of a triplex-forming peptide nucleic acid (PNA) to the transport peptide, antennapedia (Antp), we achieved successful in vivo chromosomal genomic modification of hematopoietic progenitor cells, while still retaining intact differentiation capabilities. Following systemic administration of PNA-Antp conjugates, sequence-specific gene modification was observed in multiple somatic tissues as well as within multiple compartments of the hematopoietic system, including erythroid, myeloid, and lymphoid cell lineages. As a true functional measure of gene targeting in a long-term renewable HSC, we also demonstrate preserved genomic modification in the bone marrow and spleen of primary recipient mice following transplantation of bone marrow from PNA-Antp-treated donor mice. Our approach offers a minimally invasive alternative to ex vivo gene therapy, by eliminating the need for the complex steps of stem cell mobilization and harvesting, ex vivo manipulation, and transplantation of stem cells. Therefore, our approach may provide new options for individualized therapies in the treatment of monogenic hematologic diseases such as sickle cell anemia and thalassemia.

Characterization of TCHQ-induced genotoxicity and mutagenesis using the pSP189 shuttle vector in mammalian cells

Tetrachlorohydroquinone (TCHQ) is a major toxic metabolite of the widely used wood preservative, pentachlorophenol (PCP), and it has also been implicated in PCP genotoxicity. However, the underlying mechanisms of genotoxicity and mutagenesis induced by TCHQ remain unclear. In this study, we examined the genotoxicity of TCHQ by using comet assays to detect DNA breakage and formation of TCHQ-DNA adducts. Then, we further verified the levels of mutagenesis by using the pSP189 shuttle vector in A549 human lung carcinoma cells. We demonstrated that TCHQ causes significant genotoxicity by inducing DNA breakage and forming DNA adducts. Additionally, DNA sequence analysis of the TCHQ-induced mutations revealed that 85.36% were single base substitutions, 9.76% were single base insertions, and 4.88% were large fragment deletions. More than 80% of the base substitutions occurred at G:C base pairs, and the mutations were G:C to C:G, G:C to T:A or G:C to A:T transversions and transitions. The most common types of mutations in A549 cells were G:C to A:T (37.14%) and A:T to C:G transitions (14.29%) and G:C to C:G (34.29%) and G:C to T:A (11.43%) transversions. We identified hotspots at nucleotides 129, 141, and 155 in the supF gene of plasmid pSP189. These mutation hotspots accounted for 63% of all single base substitutions. We conclude that TCHQ induces sequence-specific DNA mutations at high frequencies. Therefore, the safety of using this product would be carefully examined.

Non-B DNA-forming Sequences and WRN Deficiency Independently Increase the Frequency of Base Substitution in Human Cells

Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.

Mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts

Adozelesin is a synthetic analog of the antitumor antibiotic, CC-1065, which alkylates N3 of adenine in the minor DNA groove in a sequence-specific manner. Here we tested the mutation spectra induced by adozelesin in the supF gene of human XP-A fibroblasts using a shuttle vector assay. Adozelesin primarily induces mutations via an A --> T transversion and a single base insertion. The A --> T transversion (43/59) was observed at the adenine alkylation site in the 5'-ATTTA* sequence (A* is the site of alkylation). The single base insertion (5/59) was observed at the 3'-side of the covalently modified adenine in the 5'-CTAAA* sequence. The results of this study suggest that the DNA alkylating sequence of adozelesin influences the type of DNA mutation.

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C-->T:A and A:T-->C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases eta and zeta by siRNAs enhanced the G:C-->T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases eta and zeta, suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase eta slightly decreased the A:T-->C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases eta and zeta are involved in the bypass of 8-OH-Gua in human cells.

Suppression of mutagenesis by 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate) by human MTH1, MTH2, and NUDT5

To assess the functions of the three human MutT-type enzymes, MTH1, MTH2, and NUDT5, mutation induction by an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP; 7,8-dihydro-8-oxo-2′-deoxyguanosine 5′-triphosphate), was examined using human 293T cells treated with their specific siRNAs. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. Escherichia coli cells were transformed with the DNAs replicated in the treated cells. The knockdown of the MTH1, MTH2, and NUDT5 proteins increased the A:T → C:G substitution mutations induced by 8-OH-dGTP. In addition, the increase in the induced mutation frequency was more evident in the triple-knockdown cells. These results indicate that all three of the human MTH1, MTH2, and NUDT5 proteins act as a defense against the mutagenesis induced by oxidized dGTP.

Roles of specialized DNA polymerases in mutagenesis by oxidized guanine

To assess the involvement of specialized DNA polymerases in the mutagenesis induced by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), shuttle plasmid DNA containing the supF gene and 8-OH-dGTP were introduced into human 293T cells in which specialized DNA polymerases were knocked-down by siRNAs. The knock-downs of DNA polymerases eta and zeta, and REV1 reduced the A:T-->C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A. In contrast, the knock-down of DNA polymerase tau did not affect the mutations induced by 8-OH-dGTP. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by 8-OH-dGTP.

Arsenite enhances the benzo[a]pyrene diol epoxide (BPDE)-induced mutagenesis with no marked effect on repair of BPDE-DNA adducts in human lung cells

Arsenite effects on the benzo[a]pyrene diol epoxide (BPDE)-DNA adduct-induced mutation were evaluated in three human lung cell-lines – A549 (wild-type p53), WI38-VA13 (p53 inhibited by SV40 large-T antigen), and H1299 (p53-null) – by using the pSP189 shuttle vector, which carries a mutation target supF gene. Arsenite alone had no significant effect on the spontaneous supF mutation. BPDE modification of pSP189 enhanced the mutation rates of supF 4.37-fold, 2.96-fold, and 1.95-fold for A549, WI38-VA13, and H1299, respectively. Arsenite potentiated the BPDE-induced mutation rates of supF 2.30-fold, 2.31-fold, and 2.35-fold in A549, WI38-VA13, and H1299, respectively. These results suggest that arsenite potentiates the BPDE-induced supF mutation via a p53-independent mechanism. By using the host cell reactivation assay, we evaluated arsenite effect on repair of BPDE-DNA adducts. We found that the arsenite treatments resulting in relative survival rates ⩾65% had no significant effect on repair of BPDE-DNA adducts, indicating that p53 status did not significantly affect the repair of BPDE-DNA adducts. This study reveals that arsenite enhances the BPDE-DNA adduct-induced mutagenesis with no marked effect on repair of BPDE-DNA adducts, suggesting that arsenic may act as a co-mutagen to promote the development of human lung cancer.

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.

Mutagenicity and Sequence Specificity of Acrolein-DNA Adducts

Acrolein (Acr) is a major toxicant in cigarette smoke (CS); it can interact with DNA forming two major adduct isomers: alpha-OH-Acr-dG and gamma-OH-Acr-dG. Previously, we found that the Acr-DNA binding pattern in the human p53 gene coincides with the p53 mutational pattern in CS-related lung cancer; hence, we proposed that Acr is a major lung cancer etiological agent [ Feng , Z. , Hu , W. , Hu , Y. , and Tang , M.-s. ( 2006 ) Acrolein is a major cigarette-related lung cancer agent: Preferential binding at p53 mutational hotspots and inhibition of DNA repair . Proc. Natl. Acad. Sci. U.S.A. 103 , 15404 - 15409 ]. This hypothesis has been brought into question with recent work that failed to detect Acr-induced mutations in the pSP189 system [ Kim , S. I. , Pfeifer , G. P. , and Besaratinia , A. ( 2007 ) Lack of mutagenicity of acrolein-induced DNA adducts in mouse and human cells . Cancer Res. 67 , 11640 - 116472 ]. To resolve this controversy, we determined the level and the type of Acr-dG formation, and the mutagenicity of Acr-dG adducts in the same pSP189 system. We also mapped the Acr-dG adduct distribution at the nucleotide level and the Acr-dG-induced mutational spectrum in this system. We found that (1) gamma-OH-Acr-dG is the major adduct formed in Acr-modified DNA based on the LC-ESI-MS/MS analysis; (2) the mutation frequency is proportional to the extent of Acr modifications, the majority of which are G:C to T:A and G:C to A:T mutations; and (3) sequences with a run of Gs are the mutational hotspots. Using the UvrABC nuclease incision method to map the Acr-dG distribution in the supF gene sequence, we confirmed that Acr-DNA adducts preferentially form in guanine-rich sequences that are also mutational hotspots. These results reaffirm that Acr-dG adducts are mutagenic and support our hypothesis that Acr is a major etiological agent for CS and cooking fume-related lung cancer.

Mutagenic Specificity of N-Nitrosotaurocholic Acid in supF Shuttle Vector Plasmids

The mutagenic specificity of N-nitrosotaurocholic acid (NO-TCA) in human cells was investigated using supF shuttle vector plasmids. The plasmids pMY189 were treated with NO-TCA in vitro and introduced into normal fibroblasts (WI38-VA13) and nucleotide excision repair (NER)-deficient cells (XP2OS(SV)) for replication. The background mutation frequency of the supF gene was 4.1× 10-4 and 2.0×10-4 after replication in normal and NER-deficient cells, respectively. The mutation frequency increased 5 and 15 times in normal and NER-deficient cells, respectively, after the treatment of the plasmid with 50 mg/mL of NO-TCA. The higher mutation frequency in NER-deficient cells indicates that the DNA damage induced by NO-TCA is repaired by NER. Base sequence analysis of 101 and 94 plasmids with mutations in the supF gene propagated in normal and NER-deficient cells, respectively, revealed that the majority of the mutations were base substitutions (about 89 and 90%) and the rest were deletions and insertions (about 11 and 10%) in both cell lines. About half of the mutant plasmids contained a single base substitution. Of the single base substitutions, the most frequent mutations were G:C to A:T transitions (about 37 and 36%), followed by G:C to C:G transversions (about 31 and 28%) in both cell lines. The mutations were not distributed randomly but were located at several hot spots in the supF gene, and almost all hot spots were at G:C sites. These observations accord with previous findings that NO-TCA forms DNA adducts with dC and induces G:C to A:T base substitution in Salmonella typhimurium TA100.

Mutagenicity of tamoxifen DNA adducts in human endometrial cells and in silico prediction of p53 mutation hotspots

Tamoxifen elevates the risk of endometrial tumours in women and α-(N2-deoxyguanosinyl)-tamoxifen adducts are reportedly present in endometrial tissue of patients undergoing therapy. Given the widespread use of tamoxifen there is considerable interest in elucidating the mechanisms underlying treatment-associated cancer. Using a combined experimental and multivariate statistical approach we have examined the mutagenicity and potential consequences of adduct formation by reactive intermediates in target uterine cells. pSP189 plasmid containing the supF gene was incubated with α-acetoxytamoxifen or 4-hydroxytamoxifen quinone methide (4-OHtamQM) to generate dG-N2-tamoxifen and dG-N2-4-hydroxytamoxifen, respectively. Plasmids were replicated in Ishikawa cells then screened in Escherichia coli. Treatment with both α-acetoxytamoxifen and 4-OHtamQM caused a dose-related increase in adduct levels, resulting in a damage-dependent increase in mutation frequency for α-acetoxytamoxifen; 4-OHtamQM had no apparent effect. Only α-acetoxytamoxifen generated statistically different supF mutation spectra relative to the spontaneous pattern, with most mutations being GC→TA transversions. Application of the LwPy53 algorithm to the α-acetoxytamoxifen spectrum predicted strong GC→TA hotspots at codons 244 and 273. These signature alterations do not correlate with current reports of the mutations observed in endometrial carcinomas from treated women, suggesting that dG-N2-tam adduct formation in the p53 gene is not a prerequisite for endometrial cancer initiation in women.

Mutation spectra induced by 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide in the supF gene of human XP-A fibroblasts

1-Nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide are oxidative metabolites that are responsible for the mutagenicity of 1-nitropyrene. In this study, the mutation spectra induced by oxidative metabolites in human cells were determined using a shuttle vector assay. The mutation frequencies induced by 1-nitropyrene 9,10-oxide were 2-3 times higher than those induced by 1-nitropyrene 4,5-oxide. The base substitutions induced by 1-nitropyrene 4,5-oxide were G --> A transitions, G --> C transversions, and G --> T transversions. In the case of 1-nitropyrene 9,10-oxide, G --> A transitions, G --> T transversions, A --> G transitions and G --> C transversions were observed. Most base substitution mutations induced by oxidative metabolites occurred at the guanine sites in the supF gene. These sequence-specific hot spots were commonly identified as 5'-GA sequences for both metabolites. On the other hand, the sequence-specific hot spots at the adenine sites were identified as 5'-CAC sequences for 1-nitropyrene 9,10-oxide. These results suggest that the oxidative metabolites of 1-nitropyrene induce sequence-specific DNA mutations at the guanine and adenine sites at high frequency.

Processing of triplex-directed psoralen DNA interstrand crosslinks by recombination mechanisms

Gene targeting via homologous recombination (HR) is an important application in biotechnology and medicine. However, in mammalian cells HR is much less efficient than random integration. Triplex-forming oligonucleotides (TFOs) linked to DNA damaging agents (e.g. psoralen) can stimulate HR, providing the potential to improve gene therapy applications. To elucidate factors affecting TFO-directed psoralen interstrand crosslink (ICL)-induced recombination, we constructed a series of plasmids with duplicated supF reporter genes, each containing an inactivating deletion, to measure HR frequencies in mammalian cells. Our results indicated that TFO-directed ICL-induced recombination frequencies were higher in the plasmids with larger distances between duplicated supF genes than with a smaller separation distance. However, the position of the ICL relative to the reporter genes did not affect HR frequencies. Recombination spectra were altered by the distance between supF copies. Although single-strand annealing (SSA) recombinants were predominant in all plasmid substrates, the plasmid with the shortest interval (60 bp) revealed a significant proportion of gene conversions (GCs). GCs occurred exclusively in the gene containing the shortest deletion, regardless of the distance between supF genes, ICL position or deletion orientation. Our analyses indicated that SSA is the predominant mechanism of ICL processing of these substrates in mammalian cells.

Transcription-coupled repair pathway in UVC-induced SupF gene mutation in Tet-on 293 cell line

Objective To explore the role of transcription-coupled repair (TCR) pathway in the UVC-induced SupF gene mutation in Tet-on 293 cell line, we designed and constructed a Tet-responsive plasmid DNA pTCR-C1, and utilized this pTCR-C1 plasmid to obtain the mutation frequency of SupF reporter gene induced by UVC in Tet-on 293 cell line. Methods SupF gene was cloned into a Tet-responsive plamid pBI-L, which include a bidirectional Tet-responsive promoter, and was named pTCR-C1. The pTCR-C1 plasmid was transfected into Tet-on 293 cell line, and the mutation frequency of SupF reporter gene was detected in the presence and absence of DOX. Results The pTCR-C1 plasmid was identified with the methods of restriction digestion and DNA sequencing. The mutation frequency of SupF reporter gene in the presence of DOX was higher than in the absence of DOX. Conclusion The TCR pathway takes part in the UVC-induced SupF gene mutation in Tet-on 293 cell line.

Translesion synthesis of 7,8-dihydro-8-oxo-2′-deoxyguanosine by DNA polymerase eta in vivo

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) is one of the most common DNA lesions induced by oxidative stress. This lesion can be bypassed by DNA polymerase eta (Pol η) using in vitro translesion synthesis (TLS) reactions. However, the role that Pol η plays in vivo contributing to 8-oxo-dG mutagenesis remains unclear. To clarify the role of Pol η in 8-oxo-dG mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector (pSP189) which replicates in mammalian cells. The pSP189 plasmid was treated with methylene blue plus light (MBL), which produces predominantly 8-oxo-dG in DNA, and was then replicated in GM637 cells in presence of siRNA that knocks down the expression of Pol η, or in XP-V cells, which lack functional Pol η. The mutant frequencies were increased in the Pol η siRNA knockdown cells and in XP-V cells relative to control, meaning that Pol η plays an important role in preventing 8-oxo-dG mutagenesis. In the same system, knockdown of OGG1 also led to an increase in mutagenesis. Neither the type of mutations nor their distribution along the supF gene were significantly different between control and target specific siRNA-transfected cells (or XP-V cells) and were predominantly G to T transversions. These results show that Pol η has an important role in error-free 8-oxo-dG lesion bypass and avoidance of oxidative stress-induced mutagenesis in vivo.

MEN1 and FANCD2 mediate distinct mechanisms of DNA crosslink repair

Cells mutant for multiple endocrine neoplasia type I (MEN1) or any of the Fanconi anemia (FA) genes are hypersensitive to the killing effects of crosslinking agents, but the precise roles of these genes in the response to interstrand crosslinks (ICLs) are unknown. To determine if MEN1 and the FA genes function cooperatively in the same repair process or in distinct repair processes, we exploited Drosophila genetics to compare the mutation frequency and spectra of MEN1 and FANCD2 mutants and to perform genetic interaction studies. We created a novel in vivo reporter system in Drosophila based on the supF gene and showed that MEN1 mutant flies were extremely prone to single base deletions within a homopolymeric tract. FANCD2 mutants, on the other hand, had a mutation frequency and spectrum similar to wild type using this assay. In contrast to the supF results, both MEN1 and FANCD2 mutants were hypermutable using a different assay based on the lats tumor suppressor gene. The lats assay showed that FANCD2 mutants had a high frequency of large deletions, which the supF assay was not able to detect, while large deletions were rare in MEN1 mutants. Genetic interaction studies showed that neither overexpression nor loss of MEN1 modified the ICL sensitivity of FANCD2 mutants. The strikingly different mutation spectra of MEN1 and FANCD2 mutants together with lack of evidence for genetic interaction between these genes indicate MEN1 plays an essential role in ICL repair distinct from the Fanconi anemia genes.

Lack of Mutagenicity of Acrolein-Induced DNA Adducts in Mouse and Human Cells

Acrolein is an endogenous metabolite and a ubiquitous environmental pollutant. Recently, it has been suggested that acrolein is a major etiologic agent for tobacco smoking-related lung cancer. Despite the known DNA-damaging effects of acrolein, its mutagenicity to mammalian cells remains uncertain. We have investigated acrolein-induced DNA damage in relation to mutagenesis, with special focus on DNA repair, in mouse and human cells. We mapped the formation of acrolein-induced DNA adducts and the kinetics of repair of the induced lesions in the cII transgene, the mutational target, in acrolein-treated transgenic mouse fibroblasts. Acrolein-DNA adducts were formed preferentially at specific nucleotide positions, mainly at G:C base pairs, along the cII transgene. The induced acrolein-DNA adducts were moderately resistant to DNA repair. Quantification of cII mutant frequency in acrolein-treated cells, however, revealed that acrolein was not mutagenic to these cells at doses sufficient to produce DNA adducts. Determination of supF mutant frequency in DNA repair-proficient and DNA repair-deficient human fibroblasts transfected with acrolein-treated plasmids confirmed a lack of acrolein mutagenicity. Because CpG methylation may intensify acrolein-DNA adduction, we examined whether the extent of CpG methylation in the supF gene can determine acrolein-induced mutagenesis in human cells. Enhancement of acrolein-DNA adduction by methylating CpGs in the supF sequence did not elicit a mutagenic response in human fibroblasts, however. We conclude that acrolein is not mutagenic to mouse and human fibroblasts, regardless of DNA repair capacity or methylation status of CpGs, possibly because of a highly accurate replication bypass of the induced lesions.

Characterization of carbadox-induced mutagenesis using a shuttle vector pSP189 in mammalian cells

Carbadox, a quinoxaline 1,4-dioxide derivative, is a known mutagen with its functional mechanism yet to be well defined. In the present study we used a shuttle vector assay in vitro to uncover the functional details of carbadox-induced mutagenesis in mammalian cells. The plasmid DNA of a shuttle vector pSP189 was treated with different doses of carbadox at 37 °C for 1 or 2 h with or without the presence of S9. The target gene SupF in the plasmid was sequenced after replication in Vero cells followed by amplification in Escherichia coli MBM7070 to evaluate mutation frequency. DNA sequencing analysis of recovered carbadox-induced mutations revealed 76.3% single base substitution, 7.9% single base insertion, 10.5% single base deletion and 5.3% large fragments deletion. All single base substitutions occurred at G:C base pairs, among which transversion and transition occurred at a 2:1 ratio. The mutations did not occur randomly in the supF gene, but had sequence specificity and hotspots instead: most substitutions were detected at the nucleotide N in a 5′-NNTTNN-3′ sequence; 75% of base insertions were seen in the 5′-TCC-3′ sequence; whereas all large fragments deletions occurred in the 5′-ANGGCCNAAA-3′ sequence. Nucleotide 129, 141 and 155 in the supF gene of plasmid pSP189 were identified as the hotspots for carbadox-induced mutations that accounted for 65% of all single base substitutions. We conclude that carbadox and its metabolites induce sequence-specific DNA mutations at high frequencies, therefore its safe usage in animal husbandry should be seriously considered.

AlkB Influences the Chloroacetaldehyde-Induced Mutation Spectra and Toxicity in the pSP189 supF Shuttle Vector

2-Chloroacetaldehyde (CAA), a metabolite of the carcinogen vinyl chloride, reacts with DNA to form cyclic etheno ()-lesions. AlkB, an iron-/alpha-ketoglutarate-dependent dioxygenase, repairs 1, N (6)-ethenodeoxyadenosine (A) and 3, N (4)-ethenodeoxycytidine (C) in site-specifically modified single-stranded viral genomes in vivo and also protects the E. coli genome from the toxic effects of CAA. We examined the role of AlkB as a cellular defense against CAA by characterizing the frequencies, types, and distributions of mutations induced in the double-stranded supF gene of pSP189 damaged in vitro and replicated in AlkB-proficient (AlkB (+)) and AlkB-deficient (AlkB (-)) E. coli. AlkB reduced mutagenic potency and increased the survival of CAA-damaged plasmids. Toxicity and mutagenesis data were benchmarked to levels of -adducts and DNA strand breaks measured by LC-MS/MS and a plasmid nicking assay. CAA treatment caused dose-dependent increases in A, C, and 1, N (2)-ethenodeoxyguanosine (1, N (2)-G) and small increases in strand breaks and abasic sites. Mutation frequency increased in plasmids replicated in both AlkB (+) and AlkB (-) cells; however, at the maximum CAA dose, the mutation frequency was 5-fold lower in AlkB (+) than in AlkB (-) cells, indicating that AlkB protected the genome from CAA lesions. Most induced mutations in AlkB (-) cells were G:C to A:T transitions, with lesser numbers of G:C to T:A transversions and A:T to G:C transitions. G:C to A:T and A:T to G:C transitions were lower in AlkB (+) cells than in AlkB (-) cells. Mutational hotspots at G122, G123, and G160 were common to both cell types. Three additional hotspots were found in AlkB (-) cells (C133, T134, and G159), with a decrease in mutation frequency and change in mutational signature in AlkB (+) cells. These results suggest that the AlkB protein contributes to the elimination of exocyclic DNA base adducts, suppressing the toxic and mutagenic consequences induced by this damage and contributing to genetic stability.