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Molecular and Cellular Biology, July 2005, p. 5823-5833, Vol. 25, No. 14
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.14.5823-5833.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage{dagger}

Yingying Guo,1,3,{ddagger} Linda L. Breeden,3 Helmut Zarbl,1,3 Bradley D. Preston,2 and David L. Eaton1,3*

Departmental of Environmental and Occupational Health Sciences,1 Department of Pathology, University of Washington,2 Fred Hutchinson Cancer Research Center, Seattle, Washington3

Received 7 January 2005/ Returned for modification 11 March 2005/ Accepted 25 April 2005


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ABSTRACT
 
Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in strains defective in nucleotide excision repair (NER) (rad14), postreplication repair (PRR) (rad6, rad18, mms2, and rad5), homologous recombinational repair (HRR) (rad51 and rad54), base excision repair (BER) (apn1 apn2), nonhomologous end-joining (NHEJ) (yku70), mismatch repair (MMR) (pms1), translesion synthesis (TLS) (rev3), and checkpoints (mec1-1, mec1-1 rad53, rad9, and rad17). Together our data suggest the involvement of homologous recombination and nucleotide excision repair, postreplication repair, and checkpoints in the repair and/or tolerance of AFB1-induced DNA damage in the yeast model. Rev3 appears to mediate AFB1-induced mutagenesis when error-free pathways are compromised. The results further suggest unique roles for Rad5 and abasic endonuclease-dependent DNA intermediates in regulating AFB1-induced mutagenicity.


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INTRODUCTION
 
Mutagenic substances, including many environmental contaminants, are structurally diverse and range from simple compounds such as methylating agents to bulky DNA damaging agents (57). Differences in DNA repair capacity may play an important role in determination of individual susceptibility to endogenous and exogenous mutagens (88). DNA repair and mutagenesis have been less well studied for bulky mutagens, partly because the majority of them require efficient bioactivation by biotransformation enzymes that are absent in many in vitro study models (35).

A yeast model has been established in this study to delineate the mechanisms involved in the elimination and/or tolerance of DNA damage produced by a bulky mutagen, aflatoxin B1 (AFB1). A natural toxin produced by the common fungal mold Aspergillus flavus (21), AFB1 is one of the most potent mutagens ever characterized in eukaryotes (12, 31, 62). It requires biotransformation by cytochrome P450 (CYP450)-dependent monooxygenases to the reactive, yet highly unstable AFB1-8,9-exo-epoxide (AFBO) to become toxic and carcinogenic (21). AFBO can react with the N7 guanine residue of DNA to form an unstable trans-8,9-dihydro-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct (AFB1-N7-Gua) that is thought to either be removed hydrolytically to form an abasic (AP) site or to undergo a hydrolytic reaction that opens the guanine imidazole ring, forming a stable and persistent formamidopyrimidine (AFB1-FAPY) derivative (61, 93). Studies have shown that human CYP1A2 (hCYP1A2) is the high-affinity CYP450 enzyme active at the low AFB1 concentrations typically encountered in dietary exposures (26). Consequences of AFB1-induced DNA lesions include chromosomal strand breaks, chromosomal aberrations, micronuclei (75, 93), and point mutations (24, 37).

The specific mechanisms of DNA repair and/or tolerance to AFB1-induced DNA damage are not well understood. Although nucleotide excision repair (NER) plays an important role in the repair of AFB1-DNA lesions in Escherichia coli and human cells (54, 74, 83), considerable residual capacity remains for removal of AFB1-DNA adducts in human cells deficient in NER (94). Among the other major DNA repair pathways, base excision repair (BER) has been suggested to remove AFB1-DNA adducts (74, 94). Other pathways have been shown to participate in the repair and/or tolerance of other types of DNA damage-induced single-strand breaks (SSBs) (10), double-strand breaks (DSBs) (3, 55), and replication-blocking lesions (10, 97). Cell cycle checkpoints are likely to be involved in the repair and/or tolerance process as well, due to their regulatory connections with DNA repair pathways (103).

To evaluate the participation of different DNA repair pathways in repair of AFB1-induced DNA adducts and their coordination with cell cycle checkpoints, we examined a series of isogenic Saccharomyces cerevisiae haploid mutants representative of different DNA damage response pathway deficiencies. Previous studies have found S. cerevisiae to be a robust model system to study DNA repair. Many genes involved in DNA repair and cell cycle checkpoints are conserved among yeast and higher eukaryotic cells, and mutant strains of yeast that contain specific gene deletions are available for all the known eukaryotic DNA repair pathways (25). The mutant yeast strains used in the present study were engineered to express hCYP1A2, and their sensitivities to cell killing and mutagenesis by AFB1 treatment were determined. Specifically, we focused on whether NER and other DNA repair pathways are responsible for tolerance to AFB1, what roles each of these pathway plays in the cell's response to AFB1, and how various cell cycle checkpoints are integrated to DNA repair pathways following AFB1-induced DNA damage. Our data reveal complex relationships among various DNA repair pathways and cell cycle control in AFB1-induced cytotoxicity and mutagenesis in the yeast model.


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MATERIALS AND METHODS
 
Yeast strains. All haploid strains used in the study were in the A346a background, except yku70, mms2, and apn1 apn2, which were in the S288C background (Table 1). Double mutants were constructed by standard genetic methods.


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  		TABLE 1. Saccharomyces cerevisiae strains

Both mutant and wild-type strains were transformed to express hCYP1A2 from the GAPDH promoter on a 2µm URA3+ plasmid (pHE36) (68). The same vector without the insert (pDP34) was used as a control. Plasmids were kindly provided by Christian Sengstag (Schwerzenbach, Switzerland), and their structures were described previously (22).

The Standard Transformation Protocol in Frozen-EZ Yeast Transformation II (Zymo Research, Orange, CA) was used for transformation, and transformants were cultured in minimal synthetic media lacking uracil. The expression of hCYP1A2 cDNA in various strains was confirmed by Western blots, as described previously (22, 49).

Preparation of S9 fractions. S9 fractions of yeast strains expressing hCYP1A2 or empty vector were prepared as previously described (86), except that growth conditions were modified to match those used in the cytotoxicity and mutagenicity studies. Specifically, 106 cells were grown for 7 h in 250 ml of synthetic media lacking uracil. Protein concentrations were determined using Bradford reagent (Bio-Rad, Hercules, CA) with bovine serum albumin as the standard.

Determination of methoxyresorufin O-demethylase (MROD) enzymatic activity. MROD activities of S9 fractions from various yeast strains expressing hCYP1A2 were determined by fluorescence spectrophotometry, using a 96-well plate format modified from a previous procedure (50).

Colony-forming ability and L-canavanine resistance forward mutation assays. Engineered haploid strains were grown to similar density (1 x 106 cells/ml) in uracil-deficient medium at 30°C to continually select for the hCYP1A2 plasmid. Each culture was then split into multiple 11-ml cultures and treated with AFB1 dissolved in dimethyl sulfoxide (DMSO) at final concentrations of 5, 10, 25, and 50 µM, whereas the vehicle control was treated with 4% DMSO. Only slow-growing rad6 cultures were used to avoid possible suppressor mutations in the SRS2 gene, which can readily accumulate in the rad6 strains and affect their sensitivity. All control and treated samples were shaken for 4 h at 30°C. Ten milliliters of cell suspension was taken from each condition, and cells were harvested by centrifugation, washed with phosphate-buffered saline (PBS), and resuspended in 308 µl PBS. After sonication, 5 µl of stock cell suspensions from treated and control conditions were serially diluted into 4°C sterile PBS solution to determine cell concentration using a hemocytometer. A specific volume of serial dilutions from each condition (equivalent to 350 cells) was plated in triplicate on complete yeast extract-peptone-dextrose agar plates to count viable cells. In parallel, 100-µl (1.6 x 107 to 6.4 x 107 cells) aliquots of the original stock suspension of each condition were also plated in triplicate onto yeast extract-peptone-dextrose plates without arginine but containing 60 µg/ml of L-canavanine sulfate (Sigma, St. Louis, MO) to select for forward mutation to canavanine resistance. The numbers of yeast colonies on can+ and complete medium plates were scored after 5 days or 2 days of incubation at 30°C, respectively. The cell survival rate was calculated as the number of colonies formed on complete medium plates in the treated condition relative to that in the control condition. The mutation frequency was determined as canavanine-resistant cells per 106 viable cells for each AFB1 dose used. In each strain, the mutation frequency observed in the starting control culture was adopted as the indicator of spontaneous mutation rate, and the induced mutation frequencies by AFB1 were calculated by subtracting the observed mutation frequency in the control culture from those in the treated conditions (77). In cases when the subtraction gave a low negative number, a zero value was plotted at that point as the induced events by AFB1.

Statistical analyses. One-way analysis of variance analysis in SPSS (Statistical Package for the Social Sciences) was used to compare AFB1-induced mutation frequencies between the wild type and each individual yeast mutant.


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RESULTS
 
Experimental design. Yeast mutants comprising a panel of haploids (Table 1) deficient in one or more genes involved in specific DNA repair or checkpoint pathways were transformed with hCYP1A2. Each of these pathways was previously shown to be important for cell survival and mutagenesis in response to DNA damage and together represent the major pathways implicated in the genomic instability of cancer cells. Mutants were compared with the wild-type parental strain with respect to cytotoxicity and mutagenesis after exposure to AFB1. We reasoned that if AFB1 cytolethality is mediated by its genotoxicity, then repair-deficient strains would likely exhibit enhanced susceptibility to the lethal and mutagenic effects of AFB1 relative to wild-type cells after induction of an equal amount of DNA damage. Moreover, the use of double mutants can provide insights into interaction among pathways. A synergistic effect on the level of AFB1 cytolethality in a double mutant would indicate that two disrupted genes represent alternative repair pathways that compete for common DNA lesions. If the effect of the individual mutants is additive, then the two genes are probably involved in separate pathways that process different DNA lesions (100). Evidence exists to suggest that the NER pathway could play a role in the repair of AFB1-induced DNA adducts in yeast (54, 74, 83). NER is also an effective back-up repair system for some DNA damaging agents (64). We therefore constructed double mutants that combine mutations in the NER pathway with other DNA repair pathways or checkpoints.

Characterization of hCYP1A2 expression in various yeast strains. The stability of the expression level of hCYP1A2 from the 2µm circle-based plasmid was characterized within each strain under the same growth conditions as those used in the toxicity studies. As shown in the representative Western blots (Fig. 1), the vector-only strain did not exhibit hCYP1A2 expression. Within each strain expressing hCYP1A2 (wild type, rad9, or rad14 mutant), a comparison of three subclones derived from the same transformant demonstrated that there was little clonal variation in hCYP1A2 protein expression. These results suggested that enzyme expression at the cell population level was reasonably stable within each strain under the experimental conditions used for assessing toxicity and mutagenicity.



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  		FIG. 1. Stability of hCYP1A2 expression in various yeast strains. Different subclones derived from the same clone expressing hCYP1A2 were grown under standardized conditions as described in Materials and Methods. Clonal variations of hCYP1A2 expression within each strain (wild type, rad9, or rad14) were characterized by Western blots. There were no substantial differences in the expression levels of hCYP1A2 across different subclones derived from the same clone within one strain. Vector-only expressing strain did not have hCYP1A2 expression. WT, wild-type; ctrl, control.

When cell survival and mutagenesis following AFB1 treatment in mutants is compared with that in the wild-type yeast strains, a confounding factor is the amount of DNA damage elicited by AFB1 treatment in the mutant and wild-type strains. In order to minimize this problem, only those strains with a comparable or lower level of hCYP1A2 activity relative to the corresponding wild type were chosen for further evaluation. This strategy ensured that the levels of DNA damage among the various mutants was comparable to or lower than the levels in the parental wild-type strain. As such, the data from the cytotoxicity and mutagenesis studies that followed would either be directly comparable or would underestimate the true effects of individual gene disruptions on the phenotype. Therefore, multiple clones were isolated from each mutant strain after transformation with hCYP1A2 cDNA. These clones obtained after initial transformation were screened for hCYP1A2 protein expression and enzymatic activity by Western blot analyses (data not shown) and the MROD activity assay (see Fig. S1 in the supplemental material), respectively. MROD activity in S9 fractions is a reliable and convenient assay to measure in vitro CYP1A2 activity (11). This method was chosen as a quantitative measure of the AFB1 activating capacity of hCYP1A2 within each strain. All the mutants had hCYP1A2 activity levels comparable to those seen in the corresponding wild type, except for rad6, pms1, and rad9 rad14, all of which had somewhat lower activity. MROD activity was not detectable in the vector-only strain.

Cytotoxicity and mutagenicity studies. The L-canavanine resistance forward mutation assay was utilized to measure mutations before and after AFB1 treatments. On plates supplemented with canavanine, only cells with mutations that inactivate arginine permease encoded by CAN1 can survive. This assay can detect base substitution and frameshift mutations (39), both of which are induced by AFB1 treatment. A comparison of the frequencies of spontaneous mutagenesis in each mutant strain with those of their corresponding wild-type parental strain yielded results consistent with previous observations (Table 2) (13, 16, 27, 38, 39, 56, 69, 72, 77, 84, 99, 102).


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  		TABLE 2. Spontaneous mutation frequencies in DNA repair and checkpoint deficient yeast strains and their corresponding wild types

Yeast haploid mutants were then treated with AFB1 to determine the role of different repair and checkpoint pathways in AFB1-induced cytotoxicity and mutagenicity. We detected no appreciable AFB1-induced cytotoxicity or mutagenesis in the A364a wild type lacking the plasmid containing hCYP1A2, confirming both the absence of an endogenous yeast activation pathway and the expression and functionality of the hCYP1A2 vector necessary to bioactivate AFB1 to a mutagenic metabolite. As expected, the wild-type strain expressing hCYP1A2 showed dose-dependent reductions of cell survival with concomitant dose-dependent increases in mutagenicity (Fig. 2).



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  		FIG. 2. AFB1-induced cytotoxicity (A) and mutagenicity (B) with the wild type expressing hCYP1A2 (wild type) or the vector (pDP34) only. Unless otherwise stated, the A364a wild-type strain is employed in the study. Yeast strains were grown, treated for 4 h, and plated as indicated in Materials and Methods. The cell survival rate was calculated as the number of colonies formed on complete medium plates in the treated condition relative to that in the control condition. The mutation frequency was determined as canavanine-resistant cells per 106 viable cells for each AFB1 dose used. The induced mutation frequencies by AFB1 were calculated by subtracting the observed mutation frequency in the control culture from those in the treated conditions. In cases when the subtraction gave a low negative number, a zero value was plotted at that point as the induced events by AFB1. All data were summarized from triplicate experiments, except that apn1 apn2 was from duplicate experiments and expressed as the average ± standard error. ctrl, control.

Nucleotide excision repair (NER) mutant. NER includes the ATP-dependent excision of an oligonucleotide region containing bulky DNA damage by a multienzyme complex comprised of numerous factors that also participate in gene transcription (96). RAD14 is a DNA damage binding protein (29) required for the incision step of NER but not mRNA transcription (81). The rad14-deficient cells exhibited a dose-dependent increase in both AFB1-induced cell killing and mutagenesis (Fig. 3), suggesting an important role for NER in the removal and error-free repair of AFB1-induced DNA lesions in S. cerevisiae. These findings are also consistent with the central role of NER in the high-fidelity repair of AFB1-induced DNA damage in bacterial and mammalian systems (54, 74, 83). It is therefore likely that, as in E. coli (74), NER in S. cerevisiae is capable of excising AFB1-N7-Gua adducts and FAPY adducts.



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  		FIG. 3. AFB1-induced cytotoxicity (A) and mutagenicity (B, C) in the wild type, the mutant defective in nucleotide excision repair (rad14) or recombinational repair (rad51), and the double mutant defective in both repair pathways (rad51 rad14). Cells were treated as in Fig. 2.

Homologous recombinational repair (HRR) mutants. RAD51 and RAD54 play key roles in HRR (76). Rad51 binds and hydrolyzes ATP while forming nucleoprotein filaments on single- and double-stranded DNA and performs pairing and strand exchange activities. Rad54 also has DNA-dependent ATPase activity and may significantly increase Rad51p-mediated strand exchange. However, recombination repair of DSBs, including single-strand annealing activity, may occur in the absence of RAD51 and RAD54 (40). In our study, both rad51 (Fig. 3) and rad54 (see Fig. S2 in the supplemental material) mutants showed AFB1-induced cytotoxicity and mutation frequencies that were comparable to the wild type (P > 0.05). However, loss of both NER and HRR (rad51 rad14) resulted in a more dramatic increase in cytotoxicity and induced mutagenesis relative to either single mutant (Fig. 3). These findings suggest that both repair pathways play important roles in the error-free repair of common DNA damaging agents and that loss of both pathways severely compromises high-fidelity repair mechanisms. Our results, by default, also implicate HRR in the DNA damage tolerance/bypass pathway, since loss of RAD51 or RAD54 alone did not increase AFB1-induced mutation levels, and DNA adducts in rad51 or rad54 must have been removed by other repair processes, such as NER.

Base excision repair (BER) mutant. In BER of budding yeast, specific DNA glycosylases first cleave the N-glycosyl bond of the damaged base. The resulting abasic (AP) sites are then excised by a separate AP endonuclease or by an AP lyase function inherent to the glycosylase (9). Cleavage of AP sites by AP endonucleases or AP lyases produces DNA SSBs with 5'- or 3'-blocked ends, respectively, the latter being more toxic than AP sites themselves (20). Two AP endonucleases, APN1 and APN2, have been identified in budding yeast (44, 79). Together these enzymes are responsible for the removal of abasic sites and 3'-blocked ends through their respective endonuclease and 3'-phosphodiesterase functions (9).

Following exposure to AFB1, the survival of the apn1 apn2 double mutant was comparable to that of the wild-type strain (Fig. 4). These results suggest that APN1/APN2-mediated BER does not play a significant role in the repair of lesions induced by this potent mutagen, which is consistent with a recent study in a BER-deficient (fpg mutant) E. coli strain (2). Unexpectedly, disruption of the BER pathway in budding yeast actually lowered the level of AFB1-induced mutations (Fig. 4).



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  		FIG. 4. AFB1-induced cytotoxicity (A) and mutagenicity (B) with the wild type and the mutant defective in base excision repair (apn1 apn2). Cells were treated as in Fig. 2. The apn1 apn2 mutant and its corresponding wild type are in the S288C background.

Postreplication repair (PRR) mutants. PRR is mediated by the RAD6 epistasis group and consists of two separate error-free repair processes, as well as an error-prone translesion synthesis (TLS) pathway (98). In S. cerevisiae, Rad6 associates with Rad18 to target DNA damage regions, and both are necessary for the three known subpathways (4-6). Error-free PRR is responsible for the high-fidelity resolution of stalled replication forks through the cooperation of Rad51-independent recombinational bypass, Rad6-Rad18, and Rad5-Ubc13-Mms2 complexes (10). Mms2 promotes both error-free repair subpathways (98). TLS is defined as a series of DNA polymerase activities capable of replicating damaged DNA that would otherwise block replication by high-fidelity polymerases (10). TLS involves DNA polymerase {zeta} (Pol{zeta}), a complex of two proteins (Rev3p and Rev7p) that is able to bypass several types of DNA lesions, including cyclobutane pyrimidine and abasic sites (44, 71). Cells utilizing TLS are therefore able to overcome DNA damage and proceed through the cell cycle but with a corresponding increase in mutation frequency.

As shown in Fig. 5, upon AFB1 treatment, mutants defective in the upstream pathways (rad6 and rad18) showed significantly lower cell survival and mutation frequencies. These findings suggested the involvement of PRR in tolerance of AFB1-induced DNA adducts and replication-dependent mutagenesis. Mutants of both error-free repair (rad5 and mms2) pathways also exhibited reduced cell survival, suggesting the importance of strand breaks resulting from collapse of replication forks in AFB1-induced cytotoxicity and further emphasizing the importance of error-free PRR in tolerance of AFB1-derived DNA damage. However, AFB1-induced mutations were decreased in rad5 but increased in mms2. These data suggested that Rad5 and Mms2 function differently in response to AFB1. The mutagenic pathway mutant (rev3) showed comparable cell survival but lowered mutation frequency relative to its wild type following AFB1 treatment. These results suggest that the REV3-mediated mutagenic repair pathway is involved in AFB1-induced mutagenesis. This hypothesis was confirmed by the double mutant rev3 rad14 that had a cell survival rate comparable to the rad14 mutant but a mutation level approximately the same as the rev3 mutant.



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  		FIG. 5. AFB1-induced cytotoxicity (A) and mutagenicity (B) with the wild type and the mutant defective in postreplication repair (rad6 and rad18) and its error-prone pathway (rev3) and error-free pathway (rad5 and mms2). Cells were treated as in Fig. 2. The mms2 mutant and its corresponding wild type are in the S288C background.

Nonhomologous end-joining (NHEJ) mutant. In S. cerevisiae, the Yku70/Yku80 heterodimer binds to the ends of DSBs and is essential for the end-rejoining process that is independent of terminal DNA sequence homology (55). The yku70 mutant defective in this pathway (17, 65) showed the same resistance to AFB1-induced cytotoxicity and mutation as its isogenic wild-type cells (see Fig. S3 in the supplemental material). These data did not support the hypothesis that NHEJ is an effective pathway for repair of DNA lesions resulting from AFB1 treatment in this haploid strain.

Mismatch repair (MMR) mutant. MMR repairs mispaired DNA bases. The heterodimeric complex of MLH1-PMS1 interacts with other complexes such as MSH2-MSH3 to bind mispaired bases and increase the efficiency of recognition of a mismatch by the latter complex (30). The loss of PMS1 exhibits defective MMR in repair of certain mismatch substrates (101, 102). In our study, the pms1 mutant had the same resistance to AFB1 cytotoxicity as its isogenic parent and only slightly increased mutation levels at the doses used (P > 0.05) (see Fig. S4 in the supplemental material), suggesting that Pms1 is not involved in repair of AFB1-induced DNA lesions. This notion is further supported by the double mutant pms1 rad14 that exhibited no change in survival or mutation sensitivity relative to the more sensitive single mutant (rad14) (see Fig. S4 in the supplemental material).

Checkpoint mutants. We have demonstrated previously that the rate of ongoing S phase was slowed when cells were subjected to AFB1-DNA adduction by exposure to a sublethal dose of AFB1 in S. cerevisiae, implicating the involvement of cell cycle checkpoints following AFB1-induced DNA damage (28). In budding yeast, the S-phase checkpoint that arrests cells in S phase is dependent on a signal transduction cascade involving the Mec1p and Rad53p proteins (95). Basically, DNA lesions caused by stalled replication forks or the replication of damaged DNA are first recognized during S phase. The Mec1-Ddc2 and the replication factor C (RFC)-like (Rad24-RFC2-5) complexes are then recruited to sites of damage independently of each other. The RFC-like complex would in turn load the proliferating cell nuclear antigen (PCNA)-like (Ddc1-Mec3-Rad17) complex next to the Mec1-Ddc2 kinase. These damage-induced associations enable Mec1 to phosphorylate and activate effector kinases such as Rad53 and Chk1 through specific mediators such as Rad9 and subsequently influence DNA repair, DNA replication, and cell cycle transitions (58). RAD9 and the RAD24 epistasis group (RAD17, RAD24, MEC3, and DDC1) have also been proposed to act at an early step in DNA damage recognition (23, 59, 60, 70) and participate in controlling the rate of S phase (73, 78).

Mutants defective in MEC1, RAD53, RAD9, or RAD17 were tested to elucidate the importance of these checkpoints in mediating the effects of AFB1 treatment. A modest increase in cytotoxicity over the wild type was shown in all strains (Fig. 6; see also Fig. S5 in the supplemental material), yet the AFB1-induced mutation of CAN1 to can1 was virtually eliminated by deletion of any of the four DNA damage checkpoint genes (Fig. 6; see also Fig. S5 in the supplemental material). These results suggest a requirement for DNA damage checkpoints in cell viability and mutagenic repair of AFB1-induced DNA lesions.



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  		FIG. 6. AFB1-induced cytotoxicity (A) and mutagenicity (B) with the wild type and the mutant defective in cell cycle checkpoints (mec1-1, rad9 and rad17). Cells were treated as in Fig. 2.

The coordination of checkpoints with DNA repair pathways in repair of AFB1-induced DNA damage was further investigated. Rad9 is involved in the DNA damage response through the Mec1 kinase-dependent checkpoint pathway (73). The rad9 rad14 mutant exhibited an additive increase in cytotoxicity following exposure to AFB1 but significantly lower levels of mutation than the NER-deficient strain (rad14) (Fig. 6). These results suggest that Rad9 facilitates the mutagenic repair pathway after AFB1 treatment.


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DISCUSSION
 
Utilizing a series of engineered isogenic S. cerevisiae haploid mutants that express hCYP1A2 (a cytochrome P450 that bioactivates AFB1), we investigated the underlying mechanisms of the repair and/or tolerance of AFB1-induced bulky DNA adducts and their derivatives. As has been found with other DNA damaging agents (8, 14, 91, 100), we find that a complex network of error-free and mutagenic DNA repair pathways is involved in DNA damage repair and cell survival following AFB1-induced DNA damage in S. cerevisiae.

Budding yeast deficient in NER tolerates replication-blocking lesions primarily through a Rad52-dependent sister chromatid exchange (SCE) event (46). This may be important for repair and/or tolerance to AFB1-induced DNA damage, because loss of NER and HR causes a very dramatic loss of viability (Fig. 3). Both pathways are also involved in the repair of UV damage (8). Our data are also consistent with the loss of AFB1-induced recombination in a diploid rad51 mutant transformed with human CYP1A1 (48). It could be that replication forks collapse when encountering AFB1-DNA adducts, leaving single strands whose repair is likely to be dependent on Rad51 (19). We speculate that during recombination Rad51 helps minimize the formation of intermediates for REV3 polymerase. Alternatively, Rad51 may facilitate strand exchange that subsequently provides an environment that protects the cells from DNA damage and/or mutagenesis or simply hastens the repair of the broken DNA via recombination.

The synergistic increase of mutagenicity in the rad51 rad14 double mutant (Fig. 3) also suggests that both NER and HRR act on one or more common substrates. These results are consistent with the involvement of Rad1, required in NER (81), in the repair of AFB1-induced DNA damage via recombinational repair (48). In the absence of the two repair pathways, these AFB1-induced DNA lesions are channeled into a mutagenic repair pathway mediated by REV3. The lack of AFB1-induced forward mutation frequencies (CAN1 gene) in the rev3 mutant (Fig. 5) is consistent with that possibility and mirrors previous findings with AFB1 in mammalian systems (15) and studies of other genotoxic agents, including UV (44, 53, 63, 71, 82).

Though inefficient in inserting nucleotides opposite of lesions (51), the Rev3 protein, DNA Pol{zeta}, is remarkably capable of extending distorted DNA and terminally mismatched primers made by other DNA polymerases (45). It lacks a 3' to 5' exonucleolytic proofreading activity (67) and can incorporate all four nucleotides into the daughter strand with low processivity (71). Little is known about how TLS or Pol{zeta} is regulated. There is little evidence for transcriptional regulation; transcript levels are at best only marginally increased in response to UV irradiation, and they are maintained at a constant level throughout the cell cycle (89). Because of the low level of Pol{zeta} and its potential for interfering with normal replication, it is likely that Pol{zeta} is activated and recruited specifically to stalled replication forks. Our data show that the loss of checkpoints, AP endonucleases, or Rad5 results in decreased AFB1-induced mutagenesis. It would be of interest to see if these activities affect the activity or accessibility of Pol{zeta} to AFB1-induced lesions.

Our data implicated the involvement of checkpoint genes, such as RAD9, in the regulation of mutagenic repair of AFB1-induced DNA damage (Fig. 6), consistent with a previous report using other chemical mutagens and UV (78, 80). Rad9 may function in parallel on different substrates from NER, since the double mutant rad9 rad14 had an additive relationship in cytotoxicity (Fig. 6). Furthermore, the activation of budding yeast S-phase checkpoint genes DUN1 and RFC5 correlates with an increased mutation rate in cells lacking the DNA polymerase {delta} editing function (18). In fission yeast, activation of the checkpoint transcriptionally activates and regulates a translesional polymerase (47). Taken together, these studies suggest that the checkpoint response may activate translesion synthesis to tolerate certain DNA lesions by mutagenic repair. Rad9 could be involved in loading the Rev3 polymerase, rather than the normal replicase, onto the damaged DNA template (77). Alternatively, Rad9 might be required for the transcriptional activation of translesion synthesis genes after AFB1 treatment. Transcriptional induction of a variety of genes after UV irradiation has been shown to be dependent upon checkpoint genes (1).

The lowered mutation frequency in apn1 apn2 following AFB1 treatment (Fig. 4) is unexpected. It was previously thought that the loss of AP endonucleases would lead to the accumulation of AP sites and blocking 3'-end groups, which in turn would activate checkpoints, resulting in increased mutation levels (9), as suggested by the significantly enhanced sensitivity to UV or {gamma}-radiation (99). However, we find that loss of AP endonuclease activity reduces the mutagenic effects of AFB1 almost as much as loss of the error-prone polymerase (REV3) itself. This result suggests that the AP-endonuclease-dependent intermediates are a prominent source of AFB1-induced mutations and that they are usually repaired by an error-prone pathway. In the absence of AP-endonuclease activity, AFB1 adducts are repaired by NER or another error-free pathway. Additional triple mutants (e.g., apn1 apn2 rad14) will have to be constructed to test this possibility.

Rad5 mutation partially reduces AFB1-induced mutagenesis in our study (Fig. 5). Rad5 has been proposed to be involved in error-free PRR since rad5 mutations do not affect the frequency of most damage-induced mutations (41), and a multimeric complex of Rad6, Rad18, Rad5, Ubc13, and Mms2 can form and facilitate error-free PRR (92). However, UV-induced reversion of several ochre alleles is significantly decreased in rad5 mutants in an apparently allele-specific manner (52). A Rad5 deletion was also shown to partially suppress the spontaneous mutator phenotype of mph1, a strain defective in the error-free bypass of homologous recombination (85). Therefore, in our study, it could be that an alternative error-free pathway is available to AFB1-induced damage in the absence of Rad5, whose operation would eliminate the necessity for TLS. A possible candidate for an alternative, Rad5-independent error-free pathway is the damage-tolerant polymerase {eta}, encoded by RAD30, which can bypass a number of lesions in a relatively accurate manner (32, 34, 42, 66). Alternatively, Rad5 could play a role in a subset of mutagenic repair settings. Rad5 is a single-stranded DNA-dependent ATPase (43) and is known to interact with UBC9, which conjugates the small ubiquitin-related modifier (SUMO) moiety to PCNA and other proteins (36). SUMO modification of PCNA stimulates Rev3-dependent spontaneous mutagenesis (90) and may also suppress the Rad52-dependent postreplication repair pathway (33). If Rad5 is involved in the SUMO modification of PCNA, the loss of Rad5 activity could channel more AFB1-induced DNA lesions into error-free repair pathways.

We have used yeast mutants representative of the major DNA repair and checkpoint pathways to explore the cellular response to aflatoxin B1. A model is presented in Fig. 7 that summarizes our current understanding of the roles of DNA repair and checkpoint pathways in the elimination and/or tolerance of the DNA adducts produced by AFB1 in S. cerevisiae. Early studies indicated that AFB1 is strongly recombinogenic but only weakly mutagenic in yeast (87). Our work offers an explanation of these observations, in that wild-type cells readily and faithfully repair AFB1-induced damage using homologous recombination and nucleotide excision repair, consistent with the role of recombinational repair in the repair of AFB1-induced DNA damage in another study (48). Our data suggest that, in the absence of these pathways, the mutagenesis that ensues is via the error-prone translesion synthesis pathway and requires a functional DNA damage checkpoint. We have uncovered an unexpected role for AP endonucleases and Rad5 in facilitating AFB1-induced mutagenesis, and we speculate that these activities may enhance the activity or accessibility of these lesions to the TLS pathway. A recent study of the susceptibility to AFB1-induced carcinogenesis in mice correlated changes in DNA repair activities with differences in AFB1-induced toxicity (7). The yeast model offers an opportunity to systematically analyze the relative contributions of each pathway in the resolution of these lesions. Such studies will not only facilitate the understanding of AFB1 toxicity and carcinogenicity but also help identify mechanisms of drug resistance to other mutagenic and carcinogenic chemicals that form bulky adducts with DNA.



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  		FIG. 7. Repair and/or tolerance of AFB1-induced DNA damage in S. cerevisiae. Upon AFB1 adduction to DNA, genotoxic AFB1-N7-Gua DNA adducts are generated that can be converted to FAPY adducts and AP sites. In our study, Rad14-mediated NER is suggested to play an important role in excision of these DNA adducts. Furthermore, Rad51-mediated HRR may play a key role in the error-free repair of AFB1-induced DNA lesions. The error-free PRR could also be involved in tolerance of AFB1-induced DNA lesions, possibly immediate replication blockage, and would then lead to error-free repair. Cell cycle checkpoints (such as Rad9) may have a unique role in the accommodation of DNA damage and regulation of the Rev3-mediated error-prone pathway. AP endonuclease-dependent intermediates are implicated as the DNA structure responsible for AFB1-induced mutagenesis. Rad5, a component of PRR, may participate in regulating mutagenic repair. In summary, DNA lesions may be channeled between high-fidelity and mutagenic repair pathways in our system. The dashed lines indicate the hypothesized relationships.


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ACKNOWLEDGMENTS
 
We thank Julian Simon for providing yeast strains; Christian Sengstag for providing hCYP1A2 plasmid; Kerstin Gross-Steinmeyer for advice on MROD assays; Julia Sidorova, Toshio Tsukiyama, James Vary, and Thomas Fazzio for invaluable advice on yeast work; Ed Kelly for early advice in the development of the hCYP1A2-expressing strains; and Shawna Miles and Melisa Dashiell for technical assistance on yeast sporulation and tetrad analysis.

This work was supported in part by R01ES05780 and NIEHS Center grants P30ES07033, U19ES011387, and R01GM41073 and NCI Comprehensive Center grant P30 CA15704.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA 98105-6099. Phone: (206) 685-3785. Fax: (206) 685-4696. E-mail: deaton{at}u.washington.edu. Back

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/. Back

{ddagger} Present address: Department of Genetics and Genomics, Roche Palo Alto, Palo Alto, CA 94304. Back


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Molecular and Cellular Biology, July 2005, p. 5823-5833, Vol. 25, No. 14
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.14.5823-5833.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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