Excision Repair-deficient Strains Research Articles

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation.

Ribonucleotides are frequently incorporated into DNA during eukaryotic replication. Here we map the genome-wide distribution of these ribonucleotides as markers of replication enzymology in budding yeast, using a new 5′-DNA end-mapping method, Hydrolytic End Sequencing. HydEn-Seq of DNA from ribonucleotide excision repair-deficient strains reveals replicase- and strand-specific patterns of ribonucleotides in the nuclear genome. These patterns support the role of DNA polymerases α and δ in lagging strand replication and of DNA polymerase ε in leading strand replication. They identify replication origins, termination zones and variations in ribonucleotide incorporation frequency across the genome that exceed three orders of magnitude. HydEn-Seq also reveals strand-specific 5′-DNA ends at mitochondrial replication origins, suggesting unidirectional replication of a circular genome. Given the conservation of enzymes that incorporate and process ribonucleotides in DNA, HydEn-Seq can be used to track replication enzymology in other organisms.

Preparation of efficient excision repair competent cell-free extracts from C. reinhardtii cells.

Chlamydomonas reinhardtii is a prospective model system for understanding molecular mechanisms associated with DNA repair in plants and algae. To explore this possibility, we have developed an in vitro repair system from C. reinhardtii cell-free extracts that can efficiently repair UVC damage (Thymine-dimers) in the DNA. We observed that excision repair (ER) synthesis based nucleotide incorporation, specifically in UVC damaged supercoiled (SC) DNA, was followed by ligation of nicks. Photoreactivation efficiently competed out the ER in the presence of light. In addition, repair efficiency in cell-free extracts from ER deficient strains was several fold lower than that of wild-type cell extract. Interestingly, the inhibitor profile of repair DNA polymerase involved in C. reinhardtii in vitro ER system was akin to animal rather than plant DNA polymerase. The methodology to prepare repair competent cell-free extracts described in the current study can aid further molecular characterization of ER pathway in C. reinhardtii.

Somatic cell mutations caused by 365 nm LED-UVA due to DNA double-strand breaks through oxidative damage.

Evidence is accumulating indicating that UVA (320-400 nm ultraviolet light) plays an important role in photo-carcinogenesis. UVA is thought to produce reactive oxygen species in irradiated cells through photo-activation of inherent photosensitizers, and was recently reported to cause DNA double-strand breaks (DSBs) in exposed cells. We have investigated the involvement of UVA in mutations and DNA damage in somatic cells using Drosophila melanogaster larvae. Using the Okazaki Large Spectrograph, we previously observed that longer wavelength UVA (>330 nm) was more mutagenic in post-replication repair-deficient D. melanogaster (mei-41) than in the nucleotide excision repair-deficient strain (mei-9). LED-light has recently been developed as a high-dose-rate UVA source. LED-UVA light (365 nm) was also more mutagenic in mei-41 than in mei-9. The mei-41 gene was shown to be an orthologue of the human ATR gene, which is involved in the repair of DSBs through phosphorylation of histone H2AX. In order to estimate the extent to which oxidative damage contributes to mutation, we established a new D. melanogaster strain (urate-null mutant) that is sensitive to oxidative damage and has a marker to detect somatic cell mutations. When somatic cell mutations were examined using this strain, LED-UVA was mutagenic in the urate-null strain at doses that were non-mutagenic in the urate-positive strain. In an effort to investigate the generation of DSBs, we examined the presence of phosphorylated histone H2AvD (H2AX D. melanogaster homologue). At high doses of LED-UVA (>800 kJ m(-2)), levels of phosphorylated H2AvD (γ-H2AvD) increased significantly in the urate-null strain. Moreover, the level of γ-H2AvD increased in the excision repair-deficient strain but not in the ATR-deficient strain following UVA-irradiation. These results supported the notion that the generation of γ-H2AvD was mediated by the function of the mei-41 gene. It was reported that ATR functions on DSB repair in D. melanogaster. Taken together, we propose a possible pathway for UVA-induced mutation, whereby DNA double-strand breaks resulting from oxidative stress might be responsible for UVA-induced mutation in somatic cells of D. melanogaster larvae.

A Safe Bacterial Microsyringe for In Vivo Antigen Delivery and Immunotherapy

The industrial development of active immunotherapy based on live-attenuated bacterial vectors has matured. We developed a microsyringe for antigen delivery based on the type III secretion system (T3SS) of P. aeruginosa. We applied the "killed but metabolically active" (KBMA) attenuation strategy to make this bacterial vector suitable for human use. We demonstrate that attenuated P. aeruginosa has the potential to deliver antigens to human antigen-presenting cells in vitro via T3SS with considerable attenuated cytotoxicity as compared with the wild-type vector. In a mouse model of cancer, we demonstrate that this KBMA strain, which cannot replicate in its host, efficiently disseminates into lymphoid organs and delivers its heterologous antigen. The attenuated strain effectively induces a cellular immune response to the cancerous cells while lowering the systemic inflammatory response. Hence, a KBMA P. aeruginosa microsyringe is an efficient and safe tool for in vivo antigen delivery.

Photosensitization induced by the antibacterial fluoroquinolone Rufloxacin leads to mutagenesis in yeast

Rufloxacin (RFX) is an antibacterial fluoroquinolone that exhibits UVA photosensitization properties. Photosensitization reactions lead to the formation of oxidative damage, mainly via singlet oxygen. Here we explore the phototoxic and photomutagenic potency of RFX using a panel of yeast ( Saccharomyces cerevisiae) mutants affected in different DNA repair pathways. Yeast mutants provide a sensitive tool to identify the photodamage and the DNA repair pathways that cope with it. Cell viability test at increasing dose of UVA shows that both the DNA repair deficient and wild type cells are equally sensitive to RFX-induced photosensitization, demonstrating that phototoxic effect is not due to DNA injury. Photomutagenicity of RFX is evaluated by measuring the frequency of forward Can R mutations. The mutation induction is low in wild type cells. A high increase in mutation frequency is observed in strains affected in Ogg1 gene, compared to wild type and other base excision repair deficient strains. The mutation spectrum photomediated by RFX in wild type cells reveals a bias in favour of GC > TA transversions, whereas transition and frameshift mutations are less represented. Altogether data demonstrates that 8-oxo-7,8-dihydroguanine (8-oxoGua) is by far the major DNA damage produced by RFX photosensitization, leading to mutagenesis. We also explore the role played by DNA mismatch repair, translesion synthesis and post-replication repair in the prevention of mutagenic effects due to RFX exposure. In addition, we show that most of RFX photodegradation products are not mutagenic. This study defines the phototoxic and photomutagenic properties of antibacterial RFX and point out possible unwanted side effects in skin under sunlight.

酵母細胞におけるグアニン塩基の電気化学的測定法―紫外線照射影響研究への応用―

The cellular harm or mutation results from the indirect reaction through reactive oxygen species (ROS) generated by ultraviolet (UV) ray or direct reaction (such as cyclobutane pyrimidine dimmer (CPD) and 6-4 photoproduct). The amount of intracellular guanine nucleotide irradiated by the 0-80 J/m2 UV-C (254 nm) was measured by means of cyclic voltammetry (CV). The oxidation current peak of intracellular guanine nucleotide was observed. The current peak which reflects the concentration of intracellular guanine base decreased to about 80% of initial value with 80 J/m2 of UV-irradiation. The concentration of guanine nucleotide could be estimated from the oxidation peak current in CV. Moreover, formation of 8-oxodG was estimated by the increase in the mutation frequency from UV-irradiated cells of base excision repair deficient strain. It was shown that the decrease in the peak current is due to the oxidation of the guanine base. This analytical method is useful for the elucidation of the oxidation mechanism of the cell and biological tissues.

Inhibition of spontaneous mutagenesis by vanillin and cinnamaldehyde in Escherichia coli: Dependence on recombinational repair

Vanillin (VAN) and cinnamaldehyde (CIN) are dietary antimutagens that effectively inhibit both induced and spontaneous mutations. We have shown previously that VAN and CIN reduced the spontaneous mutant frequency in Salmonella TA104 ( hisG428, rfa, Δ uvrB, pKM101) by approximately 50% and that both compounds significantly reduced mutations at GC sites but not at AT sites. Previous studies have suggested that VAN and CIN may reduce mutations in bacterial model systems by modulating DNA repair pathways, particularly by enhancing recombinational repair. To further explore the basis for inhibition of spontaneous mutation by VAN and CIN, we have determined the effects of these compounds on survival and mutant frequency in five Escherichia coli strains derived from the wild-type strain NR9102 with different DNA repair backgrounds. At nontoxic doses, both VAN and CIN significantly reduced mutant frequency in the wild-type strain NR9102, in the nucleotide excision repair-deficient strain NR11634 ( uvrB), and in the recombination-proficient but SOS-deficient strain NR11475 ( recA430). In contrast, in the recombination-deficient and SOS-deficient strain NR11317 ( recA56), both VAN and CIN not only failed to inhibit the spontaneous mutant frequency but actually increased the mutant frequency. In the mismatch repair-defective strain NR9319 ( mutL), only CIN was antimutagenic. Our results show that the antimutagenicity of VAN and CIN against spontaneous mutation required the RecA recombination function but was independent of the SOS and nucleotide excision repair pathways. Thus, we propose the counterintuitive notion that these antimutagens actually produce a type of DNA damage that elicits recombinational repair (but not mismatch, SOS, or nucleotide excision repair), which then repairs not only the damage induced by VAN and CIN but also other DNA damage—resulting in an antimutagenic effect on spontaneous mutation.

Effect of benzene and its closed ring metabolites on intrachromosomal recombination in Saccharomyces cerevisiae

Genome rearrangements, such as DNA deletions, translocations and duplications, are associated with cancer in rodents and humans, and clastogens are capable of inducing such genomic rearrangements. The clastogen benzene and several of its toxic metabolites have been shown to cause cancer in animals. Benzene is associated with leukemia and other blood related disorders in humans. Benzene and metabolites tested negative in short-term bacterial mutation assays such as the Salmonella Mutagenicity Test and the Escherichia coli Tryptophan Reversion Assay. These assays, while reliable for the detection of point-mutagenic carcinogens, are incapable of detecting DNA strand break inducing xenobiotics. The yeast DEL assay is based on intrachromosomal recombination events resulting in deletions and is very sensitive in detecting DNA strand breaks. In previous results the DEL assay detected 17 Salmonella positive as well as 25 Salmonella negative carcinogens [Bishop, Schiestl, Hum. Mol. Genet. 9 (2000) 2427–2434]. The carcinogen benzene and its metabolites including phenol, catechol, p-benzoquinone and hydroquinone induced DEL recombination. The benzene metabolite 1,2,4-benzenetriol was negative. Interestingly, p-benzoquinone induced DEL recombination at a dose 300-fold lower than any of the other metabolites, suggesting that it might be responsible for much of benzene's genotoxicity. In addition, an excision repair deficient strain was used, but no difference was detected compared to the wildtype, indicating that DNA adducts subject to excision repair were not formed by benzene or its metabolites.

Intercalation into the DNA double helix and in vivo biological activity of water-soluble planar [Pt(diimine)( N, N-dihydroxyethyl- N′-benzoylthioureato)] +Cl − complexes: A study of their thermal stability, their CD spectra and their gel mobility

The interaction of newly synthesised water-soluble planar complexes of general structure [Pt(diimine)( N, N-dihydroxyethyl- N′-benzoylthioureato)] +Cl − with DNA was investigated by means of DNA melting studies, CD spectroscopy, and DNA gel mobility studies. Addition of stoichometric amounts of [Pt(diimine)H 2L- S, O]Cl complexes to polynucleotides caused a significant increase in the melting temperature of poly(dA–dT) and calf-thymus DNA, respectively, indicating that these complexes interacted with DNA and stabilised the double helical structure. The CD spectra confirmed the relatively strong binding of three related Pt(II) complexes ([Pt(2,2′-bipyridine)H 2L- S, O]Cl, [Pt(4,4′-dimethyl-2,2′-bipyridine)H 2L- S, O]Cl, and [Pt(1,10-phenanthroline)H 2L- S, O]Cl), to DNA. Comparison with the published CD spectra of ethidium bromide/DNA complex suggests a similar intercalation mode of binding. cis-[(4,4′-di- tert-butyl-2,2′-bipyridyl) N, N-di(2-hydroxyethyl)- N′-benzoylthioureatoplatinum(II)] chloride, with its very bulky tert-butyl groups, did not intercalate into the polynucleotide double helix. In DNA mobility studies in the presence of the four [Pt(diimine)H 2L- S, O]Cl complexes, only [Pt(2,2′-bipyridine)H 2L- S, O]Cl affected the DNA mobility to any detectable extent. Finally, in vivo studies on the biological activity of the complexes, using an Escherichia coli DNA excision repair deficient uvrA mutant strain, indicated that only the [Pt(2,2′-bipyridine)H 2L- S, O]Cl complex showed significant cellular toxicity and that this was, in part, linked to DNA damage.

Structure, Flexibility, and Repair of Two Different Orientations of the Same Alkyl Interstrand DNA Cross-Link

Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.

Influence of Caffeine on the Repair of Mitomycin C Induced DNA Damage

Caffeine potentiated MMC induced lethality in Salmonella typhimurium strains proficient in excision and recombinant repair but not in excision or recombinant repair deficient strains. Caffeine was found to inhibit the removal of interstrand DNA crosslinks induced by MMC in strains proficient for excision repair but not in a strain deficient in excision repair. Our results indicate that the potentiation by caffeine of MMC induced lethality in S. typhimurium is primarily due to the inhibition by caffeine of excision repair of interstrand DNA crosslinks.

Photoreactivation of UV-induced cyclobutane pyrimidine dimers in the MFA2 gene of Saccharomyces cerevisiae.

Photoreactive repair (PR) of cyclobutane pyrimidine dimers (CPDs) was mapped at nucleotide resolution in nucleotide excision repair (NER) proficient and deficient strains for the transcriptionally active and inactive MFA2 gene. Enhanced PR in the control region occurred in areas where no nucleosomes were present, particularly linker regions in the alpha mating type. The presence of excision plus transcriptional activation alleviated this preference, causing repair in the regions that were linker and core in the alpha mating type to be the same in this strain. Transcription had no effect on photoreactive repair in transcribed and downstream regions of MFA2, where similar rates were observed for specific CPDs in both strands. The presence of positioned nucleosomes in alpha mating types revealed slow repair in the nucleosome core, with faster repair occurring at the 3' and 5' edges. These data support the evidence that CPDs are repaired quicker in nucleosome-free regions and at edges of nucleosomes. CPDs in the linker regions are repaired more efficiently in the transcriptionally inactive strains, suggesting that nucleosome movement associated with transcription of MFA2 hampers PR irrespective of the strand. Proficient NER influenced PR in the TATA and Mcm1 binding sites by enhancing it, particularly when transcription was activated.

Inverse correlation between p53 protein levels and DNA repair efficiency in human fibroblast strains treated with 4-nitroquinoline 1-oxide: evidence that lesions other than DNA strand breaks trigger the p53 response.

Ionizing radiation-induced stabilization and the resultant transient accumulation of the p53 tumor suppressor protein is impaired in cells from ataxia telangiectasia (AT) patients, indicating a key role for ATM, the gene mutated in AT, upstream in the radiation-responsive p53 signaling pathway. Activation of this pathway is generally assumed to be triggered by DNA strand breaks produced directly following genotoxic stress or indirectly during excision repair of DNA lesions. The aim of this study was to identify the triggering signal for induction of p53 in diploid human dermal fibroblasts treated with 4-nitroquinoline 1-oxide (4NQO), a model environmental carcinogen that produces both DNA strand breaks (like ionizing radiation) and alkali-stable bulky DNA lesions (like UV light). 4NQO treatment of fibroblasts cultured from normal and AT donors and those from patients with the UV-hypersensitivity disorder xeroderma pigmentosum (XP, complementation groups A, E and G) resulted in up-regulation of p53 protein. In normal fibroblasts, there was no temporal relationship between the incidence of DNA strand breaks and levels of p53 protein; >90% of strand breaks and alkali-labile sites were repaired over 2 h following treatment with 1 microM 4NQO, whereas approximately 3 h of post-treatment incubation was required to demonstrate a significant rise in p53 protein. In contrast, exposure of normal fibroblasts to gamma-rays resulted in a rapid up-regulation of p53 and the level peaked at 2 h post-irradiation. XP cells with a severe deficiency in the nucleotide excision repair pathway showed abnormally high levels of p53 protein in response to 4NQO treatment, indicating that lesions other than incision-associated DNA strand breaks trigger p53 up-regulation. We observed a consistent, inverse correlation between the ability of the various fibroblast cultures to induce p53 following 4NQO treatment and their DNA repair efficiencies. Treatment with 0.12 microM 4NQO, for example, caused a >2-fold up-regulation of p53 in excision repair-deficient (AT, XPA and XPG) strains without eliciting any effect on p53 levels in repair-proficient (normal and XPE) strains. We conclude that up-regulation of p53 by 4NQO is mediated solely by an ATM-independent mechanism and that the p53 response is primarily triggered by persistent alkali-stable 4NQO-DNA adducts.

2,4-Nonadienal and benzaldehyde bioantimutagens in Fushimi sweet pepper (Fushimi-togarashi).

Fushimi sweet pepper, "Fushimi-togarashi", is one of the "Kyo-yasai", traditional vegetables, in Kyoto, Japan. The chloroform fraction of Fushimi sweet pepper showed bioantimutagenicity on UV induced mutation in Escherichia coli B/r WP2. The bioantimutagen was purified with silica gel chromatography and identified as 2, 4-nonadienal (ID(50) = 20 microg/plate) on the basis of GC retention time and EI-MS spectrum of authentic 2,4-nonadienal. The sweet pepper also contained a known bioantimutagen, benzaldehyde (ID(50) = 2 mg/plate). Additive bioantimutagenicity was also observed by 2, 4-nonadienal with benzaldehyde. 2,4-Nonadienal did not show bioantimutagenicity in an UV excision repair deficient strain, E. coli B/r WP2s uvrA(-)(). Furthermore no delay of the first cell division after UV irradiation was observed in E. coli B/r WP2. These results indicate that the bioantimutagenic activity of 2, 4-nonadienal on UV mutagenesis might depend on the excision repair system in E. coli B/r WP2.

Food-derived heterocyclic amines potentiate the mutagenicity of a drinking water mutagen 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX)

We investigated the enhancing effect of heterocyclic amines on base-substitution mutations with 3-amino-1,4-dimethyl-5 H-pyrido[4,3- b]indole (Trp-P-1) and 2-amino-3,4-dimethyl-imidazo[4,5- f]quinoline (MeIQ). We compared the mutagenicity of 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5 H)-furanone (MX) in the presence and absence of the heterocyclic amines in E. coli WP2 ( trpE) and in excision repair-deficient strains WP2s ( uvrA, trpE) and ZA500 ( uvrA, rfa, trpE). Since the assay was performed without microsomal metabolic activation, Trp-P-1 and MeIQ alone were not mutagenic. In WP2, trp + reversions induced by MX were greatly potentiated by Trp-P-1 and slightly potentiated by MeIQ. Mutation enhancement was not observed in strains WP2s and ZA500, suggesting that a functional DNA excision repair system is necessary for the combined action of MX and heterocyclic amines. Our finding implies that the combined effect of mutagens as well as the effect of individual mutagens, should be considered in risk evaluation.

Sensitivity to nitrogen mustard in Saccharomyces cerevisiae is independently determined by regulated choline permease and DNA repair

Sensitivity of yeast cells to the bifunctional alkylating agent nitrogen mustard (HN2) dependes on two independently operating physiological mechanisms of cellular metabolism: dynamics of uptake of HN2 via choline permease, encoded in the gene HNM1/CTR, and repair of HN2-induced DNA damage. Uptake of choline and HN2 is impaired in mutant alleles of HNM1/CTR, leading to a HN2 hyper-resistant phenotype. Overexpression of HNM1/CTR leads to HN2 sensitivity higher than that of the wild type. While mutation and regulation of HNM1/CTR have pronounced effects on the cell's HN2 sensitivity, they do not interfere with repair of HN2-induced DNA damage, a process whose quality independently determines a yeast cell's sensitivity to HN2. Consequently, HNM1/CTR overexpression in an excision repair-deficient strain leads to extreme HN2 senstivity whereas a mutational block of HNM1/CTR, in combination with excision proficiency, yields a HN2 hyper-resistant phenotype.

Temperature-dependent antimutagenic activity of acrolein in Escherichia coli

The effect of temperature on the antimutagenic activity of acrolein was investigated using UV-irradiated E. coli B. When incubated at lower temperatures (30°C or 37°C), acrolein greatly reduced the mutation frequency in WP2 (wild-type strain), but no such effect was observed with WP2s and ZA159 (excision repair-deficient strains). The antimutagenic activity of acrolein increased when cells were incubated at higher temperatures (40°C or 42°C). Particularly in excision repair-deficient strains, the antimutagenic activity was observed only at higher temperatures. In heat shock response-deficient background, however, the antimutagenic activity was observed at 30°C even in the excision repair-deficient strains.

Bio-antimutagenic activities of vitamin B 6 in E. coli and mouse peripheral blood cells

Pyridoxal (PL) and pyridoxal 5′-phosphate (PLP) showed a marked bio-antimutagenic effect on UV-induced mutagenesis in E. coli B/r WP2, but not in the DNA excision repair-deficient strain WP2s uvrA under the condition where no cellular toxicity was observed. No delay in the first cell division was seen on post-treatment with PL after UV irradiation. PL reduced not only UV- but 4-nitroquinoline-1-oxide-induced mutation, while it was ineffective in N-methyl- N′-nitro- N-nitrosoguanidine- or γ-ray-treated cells. These results suggest that PL promotes DNA excision repair directly or indirectly and the decrease in the amount of unrepaired DNA damage might cause the reduction of UV-induced mutations in E. coli B/r WP2. In addition to the above observation, PLP reduced the frequency of mitomycin C- (2 mg/kg, i.p.) induced micronuclei in mouse peripheral blood cells. Simultaneous or subsequent oral administration of PLP (25 mg/kg) decreased the frequency of micronucleated peripheral reticulocytes.

Sites of preferential induction of cyclobutane pyrimidine dimers in the nontranscribed strand of lacI correspond with sites of UV-induced mutation in Escherichia coli

An approach utilizing fluorescence-activated DNA sequencing technology was used to study the position and frequency of UV-induced lesions in the lacI gene of Escherichia coli. The spectrum of sites of UV damage in the NC+ region of the gene was compared with a published spectrum of UV-induced mutation in lacI (Schaaper, R.M., Dunn, R.L., and Glickman, B.W. (1987) J. Mol. Biol. 198, 187-202). On average, the frequency of UV-induced lesions in the nontranscribed strand was higher than that in the transcribed strand in the region analyzed. A large fraction of mutations occurs at sites of UV-induced lesions in the nontranscribed strand, but not in the transcribed strand. This bias is reduced in an excision repair deficient (UvrB-) strain. In addition, mutations occur overwhelmingly at sites where a dipyrimidine sequence is present in the nontranscribed strand. This bias is also markedly reduced in the UvrB- strain. In light of recent work Mellon and Hanawalt (Mellon, I., and Hanawalt, P.C. (1989) Nature 342, 95-98) describing the preferential removal of cyclobutane dimers from the transcribed strand of the expressed lacZ gene in E. coli, our data suggest that preferential strand repair may have a significant effect on mutagenesis.

Equivalence of UDS responses for established cell lines and primary cells derived from the mei-9a and mus201D1 excision repair-deficient strains of Drosophila melanogaster

Autoradiographic analysis of unscheduled DNA synthesis in response to DNA damage produced by relatively high doses of the direct-acting, monofunctional alkylating agent, MMS, (4.5 mM) and UV (40 J/m 2) demonstrates that established cell lines, derived from the mei-9 a and mus201 D1 excision repair-deficient strains of Drosophila melanogaster, perform no measurable incorporation of [ 3H]thymidine into repair patches, in accordance with the observations made for the corresponding primary embryonic cells derived from the two mutagen-sensitive strains of flies. These established cell lines can therefore be used as appropriate models for both examination of the biochemical basis of the genetic defects in the mei-9 and mus201 mutations and the role of excision-repair processes in spontaneous and induced mutation induction in eukaryotic cells.