Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, SK S7N 5E5 Canada
Received March 25, 2003; accepted May 22, 2003
ABSTRACT
The RNR3-lacZ genotoxicity testing system was developed based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene in response to a broad range of DNA-damaging agents. In order to enhance the sensitivity of the RNR3-lacZ system, several deletion mutant strains representing different repair pathways were created and examined for their effects on RNR3-lacZ expression. It was found that inactivation of different DNA repair pathways has profound effects on the DNA damage induction of RNR3 expression. Although deletion of MAG1 in the base excision repair pathway enhances the detection sensitivity to DNA-alkylating agents, and deletion of RAD2 in the nucleotide excision repair pathway enhances the detection sensitivity to ultraviolet and agents that produce bulky lesions, inactivation of genes involved in the recombination repair and postreplication repair variably reduces RNR3-lacZ induction. This study not only helps to establish a more sensitive genotoxicity testing system but also suggests that certain eukaryotic DNA repair pathways are required for gene regulation in response to DNA damage and probably serve as sensors in the signal transduction cascade.
Key Words: RNR3-lacZ; gene regulation; genotoxicity test; sensitivity; DNA repair; eukaryotes.
The RNR3-lacZ genotoxicity testing system based on the induction of a Saccharomyces cerevisiae RNR3-lacZ reporter gene has been developed recently (Jia et al., 2002). It is a time- and cost-saving testing system with characteristics of high sensitivity, ease of assay, and use of a eukaryotic microorganism. All tested DNA-damaging agents and agents that interfere with DNA synthesis produced positive signals in this system, whereas non-DNAtargeting agents were negative, which correlates with the Ames test very well. Based on the comparison of minimum response dose with several chemical agents, the sensitivity of the RNR3-lacZ system is comparable to or even higher than that of the bacterial SOS-ChromoTest (Jia et al., 2002
). These encouraging preliminary observations prompted us to improve the RNR3-lacZ induction sensitivity so that it can be widely accepted as a standard, short-term genotoxicity testing system complementary to mutagenesis testing systems such as the Ames test.
It has been shown that modifications of host DNA repair systems can enhance the sensitivity of both the Ames test and the SOS-ChromoTest. For example, introduction of an R factor (pKM101) into Ames strains enhanced chemical and ultraviolet (UV)-induced mutagenesis (McCann et al., 1975; Walker and Dobson, 1979
). Meanwhile, deletion of the uvrB gene, which is involved in nucleotide excision repair (NER), increased the incidence of mutagenesis and therefore lowered the threshold of sensitivity of the Ames test (Moreau et al., 1976
). Previous research in our laboratory (Xiao et al., 1996b
) also demonstrated that two potent carcinogens, 1,2-dimethylhydrazine and azoxymethane, could be detected as bacterial mutagens if Ames test strains lack the DNA repair protein O6-methylguanine methyltransferase, whereas the standard Ames test failed to detect them as mutagens (Lijinsky et al., 1985
). Similar attempts were also reported with the SOS-ChromoTest in which the introduction of a dam-3 mutation into a PQ37 derivative strain enhanced the sensitivity of the system (Quillardet and Hofnung, 1987
). The authors provided evidence that the increased SOS inducibility in the dam-3 mutant strain was specific for compounds causing DNA mismatches. Taken together, the above results suggest that, as with the Ames test and SOS-ChromoTest, the sensitivity of eukaryotic genotoxicity testing systems may be further improved by inactivating certain DNA repair pathways. Indeed, a report (Kiser and Weinert, 1996
) that deletion of RAD16 in yeast cells enhanced the sensitivity of a number of yeast DNA damage-inducible genes to DNA damage treatment supports this hypothesis. This hypothesis was systematically tested in this study with the aim of further increasing the RNR3-lacZ sensitivity to detect genotoxic agents and pollutants.
Genetic analyses and subsequent biochemical characterization have defined three major DNA radiation damage repair pathways, namely, the RAD3 NER pathway, the RAD52 recombination repair pathway, and the RAD6 postreplication repair (PRR) and mutagenesis pathway (Friedberg et al., 1995). NER predominately recognizes lesions that cause helical distortions; the recombination repair pathway is responsible for the repair of double-stranded DNA breaks; PRR is defined as an activity to convert DNA damage-induced single-stranded gaps into large molecular weight DNA without actual removal of the replication-blocking lesions, which is often referred to as a DNA damage tolerance or avoidance pathway. In addition to the above three DNA radiation damage repair pathways, genes responsible for the repair of damaged bases belong to a base excision repair (BER) pathway. The BER pathway recognizes and repairs specific base-modifying lesions that are relatively small modifications of the DNA predominantly produced by DNA-alkylating and oxidative agents (Friedberg et al., 1995
).
In this study, mutant strains defective in genes from the above four DNA repair pathways were employed as host strains to detect whether inactivation of a given DNA repair system can enhance the sensitivity of the RNR3-lacZ system. All the mutant strains were created by targeted gene deletion from a single wild type strain, allowing for comparison within the same genetic background. The expression level of RNR3-lacZ was measured in wild type and mutant strains treated with representative DNA-damaging agents. Several mutant strains were found to display enhanced inducibility of the RNR3-lacZ reporter gene in an agent-specific manner, whereas others significantly reduce RNR3-lacZ induction under the same conditions.
MATERIALS AND METHODS
Strains and plasmids.
Plasmid pZZ2 (YCp, URA3, RNR3-lacZ; Zhou and Elledge, 1992) was obtained from Dr. S. Elledge (Baylor College of Medicine, Houston, TX) and utilized to create the RNR3-lacZ testing system. All the haploid yeast strains listed in Table 1
are isogenic to the wild type strain DBY747 and were created by a one-step targeted gene deletion method (Rothstein, 1991
). To select hisG-URA3 pop-out derivatives from strains carrying the hisG-URA3-hisG allele, yeast cells grown overnight in a nonselective medium were spread on a plate containing 5-fluoroorotic acid (5-FOA; United States Biological, Swampscott, MA), and Ura- colonies were selected (Boeke et al., 1987
).
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Test chemicals and sources of radiation.
Chemicals used in this study include methyl methanesulfonate (MMS), ethyl methanesulfonate (EMS), and 4-nitroquinoline-N-oxide (4-NQO). All the above chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The 4-NQO stock solution was made in acetone at a concentration of 10 mg/ml, aliquoted, and stored at -20 °C. After treatment, yeast cells were precipitated by centrifugation, washed twice with sterile distilled water, and resuspended in Z buffer (60 mM Na2HPO4 7H2O, 40 mM NaH2PO4 H2O, 10 mM KCl, 1 mM MgSO4 7H2O, 40 mM ß-mercaptoethanol, pH 7. 0) for the ß-galactosidase (ß-gal) assay.
For UV treatment, cells were collected by centrifugation and resuspended in 100 µl sterile distilled water, plated on YPD, and exposed to 254 nm UV light in a UV cross-linker (Fisher model FB-UVXL-1000, approximately 2,400 µW/cm2) at given doses in the dark. A UV lamp (UVP model UVGL-25, 40 µW/cm2) was used when the exposure dose was lower than 10 J/m2.
For -ray irradiation, cells were collected by centrifugation and resuspended in 1 ml of sterile distilled water in a microcentrifuge tube. After the tubes containing yeast cells were exposed to a 60Co
-ray source at a dose rate of 32.1 rad/s for times calculated to achieve the desired doses, the yeast cells were diluted into SD-Ura medium and incubated at 30°C for another 4 h prior to the ß-gal assay.
Toxicity test.
Cell survival rates were determined as previously described (Jia et al., 2002). At the end of incubation and prior to the ß-gal assay, untreated and treated cells were collected by centrifugation, diluted, and plated on YPD in duplicate. The plates were incubated at 30°C for 3 days, and the number of colonies was counted. The toxic effect is expressed as a percentage of colonies from treated samples versus untreated samples.
ß-Galactosidase activity assay.
The ß-gal activity assay was performed as described previously (Xiao et al., 1993). Briefly, 0.5 ml of an overnight culture of yeast cells was used to inoculate 2.5 ml of fresh selective medium. After 2-h incubation, when cell density reached an optical density at 600 nm (OD600) of approximately 0.20.3, cells were treated with test chemicals or exposed to radiation and returned to incubation for another 4 h, which was previously found to be optimal for the RNR3-lacZ induction by a ß-gal assay (Jia et al., 2002
). One milliliter of culture was used to determine cell density at OD600. The cells from the remaining 2 ml of culture were collected by centrifugation and used for the ß-gal assay. The ß-gal activity is expressed in Miller units (Guarente, 1983
). Fold induction was calculated as a ratio of ß-gal activity of the cells with and without treatment in the same experiment. The results are the average of at least three independent experiments.
RESULTS
The overall objective of this investigation is to enhance further the sensitivity of RNR3-lacZ genotoxicity testing by surveying isogenic yeast strains defective in various DNA repair pathways. DNA-damaging agents were selected for their well-known mechanism of action and to represent different classes of agents. We felt that the relative fold induction of RNR3-lacZ before and after treatment would best reflect the ability of this system to detect genotoxicity of the testing agent, although it was previously noted that certain DNA repair mutants may display an elevated basal level activity (Hryciw et al., 2002) and, hence, undermine the fold induction. We also felt that although the detection sensitivity can be reflected by altered fold induction and the dose required to achieve maximum fold induction, a minimum dose required to achieve a 2-fold induction in a given strain is considered as a standard measurement of sensitivity, which can be determined from the graphs.
RNR3-lacZ Induction in BER Defective Strains
All BER reactions are initiated by the action of a specific class of DNA enzymes called DNA glycosylases. A glycosylase recognizes and binds to the damaged site in a lesion-specific manner and mediates the cleavage of the damaged base from the sugar backbone. MAG1 encodes a 3-methyladenine DNA glycosylase specifically involved in the repair of alkylated lesions (Chen et al., 1989). In the mag1 null mutant strain, the expression of RNR3-lacZ after MMS treatment was increased (Fig. 1A
). In particular, the maximum induction of RNR3-lacZ increased from 48-fold in wild type cells to 59-fold in the mag1 null mutant. More importantly, the optimal induction dose decreased from 0.02% in the wild type to 0.01% in the mag1 mutant, and the detection sensitivity was enhanced significantly. Hence, with MMS doses up to 0.01%, the mag1 mutant strain displays a 3- to 4-fold enhancement of induction (Fig. 1A
). This phenomenon appears to be true for other DNA-alkylating agents, because the mag1 mutant shows an enhanced sensitivity to another alkylating agent, EMS (Fig. 1B
). In this case, although the maximum fold induction is similar between wild type and mutant cells, the mag1 mutant reaches it at 0.2% EMS instead of the 0.5% in wild type cells. A 4- to 5-fold enhancement of RNR3-lacZ induction at low doses of EMS by MAG1 deletion was observed, and the response is in the linear range (Fig. 1B
). In contrast, deletion of MAG1 does not affect the RNR3-lacZ induction profile by UV treatment (Fig. 1C
) and actually decreases the RNR3-lacZ response to
-ray treatment at high doses (Fig. 1D
). These results indicate that the enhancement of RNR3-lacZ sensitivity by MAG1 deletion is probably specific to DNA-alkylating agents and limited to lesions repaired by the Mag1 DNA glycosylase.
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The abasic site generated by Mag1 DNA glycosylase is further processed by apurinic/apyrimidinic (AP) endonucleases (Seeberg, 1995) encoded by APN1 and APN2 in budding yeast; deletion of these genes results in enhanced sensitivity to killing by MMS (Bennett, 1999
; Johnson et al., 1998
) and other base-damaging agents (Bennett, 1999
). The RNR3-lacZ induction was measured in the apn1 apn2 double mutant strain after MMS treatment; to our surprise, the fold induction was actually decreased (Fig. 2
). The reduced fold induction was largely due to increased basal level RNR3-lacZ expression. One concern with MMS treatment of apn1 apn2 cells was that this mutant strain is very sensitive to killing by MMS. Deletion of the MAG1 gene in the AP endonuclease-deficient cells partially rescues the severe sensitivity to killing by MMS (Xiao et al., 2001
); however, mag1 apn1 apn2 triple mutations almost completely abolished the RNR3 induction without further increase in basal level RNR3-lacZ expression (Fig. 2
).
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4-NQO has been referred to as a UV-mimetic agent because it causes bulky lesions mainly repaired by NER (Friedberg et al., 1995). Indeed, deletion of RAD2 enhances 4-NQO induction of RNR3-lacZ by about 3-fold in the dose range examined (Fig. 3
). It is also reported that NER participates in the repair of DNA methylation damage (Xiao and Chow, 1998
; Xiao et al., 1996b
). Hence, the effect of deletion of RAD2 on the MMS-induced RNR3-lacZ expression was examined. It is interesting to notice that at low MMS doses, the rad2 mutant displayed an enhanced sensitivity. However, at doses higher than 0.1% MMS, the RNR3-lacZ induction was compromised in the rad2 mutant (Fig. 1A
). Deletion of RAD2 also compromises RNR3-lacZ induction by
-ray treatment at high doses, although no significant difference between rad2 mutant and wild type cells was observed at low doses (Fig. 1D
).
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Genetic Interactions between rad2 and mag1 with Respect to RNR3-lacZ Induction
Because the above survey of DNA repair pathways indicates that deletion of MAG1 is able to enhance the RNR3-lacZ detection sensitivity to DNA-alkylating agents, whereas inactivation of the NER pathway enhances its sensitivity to UV and UV-mimetic agents, we anticipated that a mutant carrying both mag1 and rad2 might display enhanced sensitivity to a broad range of genotoxic agents. Indeed, the mag1 rad2 double mutant is extremely sensitive to both UV and MMS (Table 2). However, with respect to RNR3-lacZ induction, the double mutant was not as sensitive to MMS as was mag1 (Fig. 1A
) and not as sensitive to UV (Fig. 1C
) and 4-NQO (Fig. 3
) as were rad2 single mutants. These results suggest a complex genetic interaction between BER and NER in the regulation of gene expression in response to environmental genotoxic agents. Synergistic interactions between mutations in these two pathways for the repair of DNA alkylation damage have been reported (Torres-Ramos et al., 2000
; Xiao and Chow, 1998
). Two possible consequences after inactivation of both BER and NER pathways would be the channeling of lesions into other repair and/or tolerance pathways and the alteration of cellular signal and signal transduction within DNA damage checkpoint cascades.
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It is known from previous studies that mutants defective in the NER pathway are more sensitive to killing by UV and UV-mimetic agents; those in the recombination pathway are more sensitive to ionizing radiations and radiation-mimetic agents such as MMS, whereas those in the PRR pathway are sensitive to a broad range of DNA-damaging agents (Broomfield et al., 2001; Friedberg et al., 1995
). In contrast, mutants defective in the BER pathway are sensitive to killing by base-damaging agents, including alkylating and oxidative agents, but do not show enhanced sensitivity to UV and radiations (Friedberg et al., 1995
; Seeberg et al., 1995
). It was also reported that inactivation of particular DNA repair pathways may reduce (Cohen et al., 2002
; Sheng and Schuster, 1993
) or enhance (Kiser and Weinert, 1996
) certain gene expression in response to DNA damage in yeast cells. In this study, the effects of inactivation of the above four major DNA repair pathways on RNR3-lacZ inducibility by representative DNA-damaging agents were investigated. Because all the mutant strains were created by a one-step targeted gene deletion from the same parental strain, the difference in the response to treatment can be attributed solely to the gene(s) under investigation. The results allow us to draw several general conclusions.
Deletion of DNA repair genes variably affects RNR3-lacZ induction by genotoxic agents. There is no clear correlation between the relative level of sensitivity and the level of induction by a given DNA repair mutant in response to a given toxic agent. For example, rad6, rad18, rad50, rad52, and apn1 apn2 mutants are more sensitive to MMS than is the mag1 mutant (Xiao et al., 1996a; Table 2
), yet only mag1 enhances RNR3-lacZ induction.
In two cases, deletion of a specific DNA repair gene significantly enhances the response of the RNR3-lacZ system to agents that cause damage primarily repaired by the corresponding pathway. This enhancement of sensitivity is potentially applicable to the improvement of the RNR3-lacZ testing systemthe objective of this study. This observation is consistent with a notion and our experimental results that lack of repair allows the accumulation of specific lesions caused by the DNA-damaging agent, which in turn signals RNR3 upregulation. Mag1 has a broad range of substrates, including lesions produced by methylating and ethylating agents, as well as other industrial alkylating agents (Berdal et al., 1998; Friedberg et al., 1995
; Matijasevic et al., 1992
). Similarly, eukaryotic NER is primarily responsible for the repair of DNA damage caused by UV, bulky DNA adducts, and DNA cross-links (Friedberg et al., 1995
). Hence, including these mutant strains in the testing system will enhance its ability to detect a wide range of genotoxic agents, especially environmental pollutants and contaminants, where the concentration of toxic agents is low.
There is a strong genetic interaction between BER and NER with respect to damage-induced RNR3-lacZ expression. In this case, the double mutant did not display a combined or further enhanced inducibility by MMS, UV, or 4-NQO. Instead, the double mutant is less sensitive than its respective single mutants to DNA damage induction. This phenomenon is in contrast to the synergistic interaction between BER and NER pathway mutations with respect to killing by MMS (Xiao et al., 1996a).
Inactivation of several genes, including APN1 and APN2 in the BER pathway and those in the recombination repair and PRR pathways, actually decreases RNR3-lacZ inducibility. This is partly due to the elevated basal-level expression in the mutant cells, indicating an increased level of endogenous DNA damage that leads to the spontaneous derepression of RNR3 gene expression. Indeed, RNR3-lacZ has been employed as a reporter of endogenous DNA damage in yeast cells (Hryciw et al., 2002). On the other hand, a common feature of the above gene products is that they recognize DNA damage intermediates instead of initial lesions caused by DNA-damaging agents. Hence, AP endonucleases recognize abasic sites derived from either glycosylase activity or spontaneous depurination (Barzilay and Hickson, 1995
); recombination repair recognizes and processes double-strand breaks mainly caused by processing of base lesions and stalled replication forks; and PRR is probably specific for single-stranded replication gaps. The above lesions may serve as DNA damage signals for the induction of RNR3 and other gene expression. Lack of sensors or the absence of proper signaling for these lesions may affect the signal transduction pathway, leading to compromised transcriptional regulation.
ACKNOWLEDGMENTS
We wish to thank Dr. Elledge for the RNR3-lacZ construct, Michelle Hanna for proofreading the manuscript, and other lab members for helpful discussion. This research was supported by the National Sciences and Engineering Council of Canada operating grant OPG0138338 to W.X.
NOTES
1 To whom correspondence should be addressed at the Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5 Canada. Fax: (306) 966-4311. E-mail: wei.xiao{at}usask.ca.
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