Amplified and distinctive genotoxicity of titanium dioxide nanoparticles in transformed yeast reporters with human cytochrome P450 (CYP) genes.

Titanium dioxide nanoparticles (nTiO2) have been considered a possible carcinogen to humans, but most existing studies have overlooked the role of human enzymes in assessing the genotoxicity of nTiO2. Here, a toxicogenomics-based in vitro genotoxicity assay using a GFP-fused yeast reporter library was employed to elucidate the genotoxic potential and mechanisms of nTiO2. Moreover, two new GFP-fused yeast reporter libraries containing either human CYP1A1 or CYP1A2 genes were constructed by transformation to investigate the potential modulation of nTiO2 genotoxicity in the presence of human CYP enzymes. This study found a lack of appreciable nTiO2 genotoxicity as indicated by the yeast reporter library in the absence of CYP expression but a significantly elevated indication of genotoxicity in either CYP1A1- or CYP1A2-expressing yeast. The intracellular reactive oxygen species (ROS) measurement indicated significantly higher ROS in yeast expressing either enzyme. The detected mitochondrial DNA damage suggested mitochondria as one of the target sites for oxidative damage by nTiO2 in the presence of either one of the CYP enzymes. The results thus indicated that the genotoxicity of nTiO2 was enhanced by human CYP1A1 or CYP1A2 enzyme and was associated with elevated oxidative stress, which suggested that the similar mechanisms could occur in human cells.

High-throughput screening of the Saccharomyces cerevisiae genome for 2-amino-3-methylimidazo [4,5-f] quinoline resistance identifies colon cancer-associated genes.

Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cigarette smoke. However, few eukaryotic resistance genes have been identified. We used Saccharomyces cerevisiae (budding yeast) to identify genes that confer resistance to 2-amino-3-methylimidazo[4,5-f] quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. Deletion libraries expressing human CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for 5 or 10 generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. We identified 424 ORFs, including 337 genes of known function, in duplicate screens of the "humanized" collection for IQ resistance; resistance was further validated for a select group of 51 genes by growth curves, competitive growth, or trypan blue assays. Screens of the library not expressing human genes identified 143 ORFs conferring resistance to IQ per se. Ribosomal protein and protein modification genes were identified as IQ resistance genes in both the original and "humanized" libraries, while nitrogen metabolism, DNA repair, and growth control genes were also prominent in the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. Among DNA Repair and checkpoint genes, we identified those that function in postreplication repair (RAD18, UBC13, REV7), base excision repair (NTG1), and checkpoint signaling (CHK1, PSY2). These studies underscore the role of ribosomal protein genes in conferring IQ resistance, and illuminate DNA repair pathways for conferring resistance to activated IQ.

The continuing evolution of barcode applications: Functional toxicology to cell lineage.

DNA barcoding is a method to identify biological entities, including individual cells, tissues, organs, or species, by unique DNA sequences. With the advent of next generation sequencing (NGS), there has been an exponential increase in data acquisition pertaining to medical diagnosis, genetics, toxicology, ecology, cancer, and developmental biology. While barcoding first gained wide access in identifying species, signature tagged mutagenesis has been useful in elucidating gene function, particularly in microbes. With the advent of CRISPR/CAS9, methodology to profile eukaryotic genes has made a broad impact in toxicology and cancer biology. Designed homing guide RNAs (hgRNAs) that self-target DNA sequences facilitate cell lineage barcoding by introducing stochastic mutations within cell identifiers. While each of these applications has their limitations, the potential of sequence barcoding has yet to be realized. This review will focus on signature-tagged mutagenesis and briefly discuss the history of barcoding, experimental problems, novel detection methods, and future directions.

CYP1B1 converts procarcinogens into genotoxins in Saccharomyces cerevisiae.

CYP1B1 activates many chemical carcinogens into potent genotoxins, and allelic variants are risk factors in lung, breast, and prostate cancer. However, few eukaryotic genetic instability endpoints have been directly measured for CYP1B1-activated metabolites. In this study, we expressed human CYP1B1 in yeast strains that measure DNA damage-associated toxicity and frequencies of chromosomal translocations. DNA damage-associated toxicity was measured in a rad4 rad51 strain, defective in both DNA excision and recombinational repair. Frequencies of chromosomal translocations were measured in diploid yeast strains containing two his3 fragments. These strains were exposed to benzo[a]pyrene-7,8-dihydrodiol (BaP-DHD), aflatoxin B1 (AFB1), and the heterocyclic aromatic amines, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). We observed that AFB1, BaP-DHD, IQ, and MeIQx conferred toxicity in the DNA repair mutant expressing CYP1B1. Translocation frequencies increased eight-fold and three-fold after exposure to 50 µM AFB1 and 33 µM BaP-DHD respectively. A DNA damage response was observed after AFB1 exposure, as measured by the induction of the small subunit of ribonucleotide reductase, Rnr3. While CYP1B1-mediated activation of BaP-DHD and heterocyclic aromatic amines was expected, activation of AFB1 to become a potent recombinagen was not expected. These studies demonstrate that chromosomal rearrangement is a useful genotoxic endpoint for CYP1B1-mediated carcinogen activation.

Genome Profiling for Aflatoxin B1 Resistance in Saccharomyces cerevisiae Reveals a Role for the CSM2/SHU Complex in Tolerance of Aflatoxin B1-Associated DNA Damage.

Exposure to the mycotoxin aflatoxin B1 (AFB1) strongly correlates with hepatocellular carcinoma (HCC). P450 enzymes convert AFB1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB1-N7-Gua) DNA adducts, which convert to stable mutagenic AFB1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB1 resistance. Here, we profiled the yeast genome for AFB1 resistance. We introduced the human CYP1A2 into ∼90% of the diploid deletion library, and pooled samples from CYP1A2-expressing libraries and the original library were exposed to 50 µM AFB1 for 20 hs. By using next generation sequencing (NGS) to count molecular barcodes, we initially identified 86 genes from the CYP1A2-expressing libraries, of which 79 were confirmed to confer AFB1 resistance. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in postreplication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes also included those functioning in checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in postreplication repair, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB1-associated sister chromatid recombination while suppressing AFB1-associated mutations. These studies thus broaden the number of AFB1 resistance genes and have elucidated a mechanism of error-free bypass of AFB1-associated DNA adducts.

Alcoholic-Hepatitis, Links to Brain and Microbiome: Mechanisms, Clinical and Experimental Research.

The following review article presents clinical and experimental features of alcohol-induced liver disease (ALD). Basic aspects of alcohol metabolism leading to the development of liver hepatotoxicity are discussed. ALD includes fatty liver, acute alcoholic hepatitis with or without liver failure, alcoholic steatohepatitis (ASH) leading to fibrosis and cirrhosis, and hepatocellular cancer (HCC). ALD is fully attributable to alcohol consumption. However, only 10-20% of heavy drinkers (persons consuming more than 40 g of ethanol/day) develop clinical ALD. Moreover, there is a link between behaviour and environmental factors that determine the amount of alcohol misuse and their liver disease. The range of clinical presentation varies from reversible alcoholic hepatic steatosis to cirrhosis, hepatic failure, and hepatocellular carcinoma. We aimed to (1) describe the clinico-pathology of ALD, (2) examine the role of immune responses in the development of alcoholic hepatitis (ASH), (3) propose diagnostic markers of ASH, (4) analyze the experimental models of ALD, (5) study the role of alcohol in changing the microbiota, and (6) articulate how findings in the liver and/or intestine influence the brain (and/or vice versa) on ASH; (7) identify pathways in alcohol-induced organ damage and (8) to target new innovative experimental concepts modeling the experimental approaches. The present review includes evidence recognizing the key toxic role of alcohol in ALD severity. Cytochrome p450 CYP2E1 activation may change the severity of ASH. The microbiota is a key element in immune responses, being an inducer of proinflammatory T helper 17 cells and regulatory T cells in the intestine. Alcohol consumption changes the intestinal microbiota and influences liver steatosis and liver inflammation. Knowing how to exploit the microbiome to modulate the immune system might lead to a new form of personalized medicine in ALF and ASH.

tRNA modification and cancer: potential for therapeutic prevention and intervention.

Transfer RNAs (tRNAs) undergo extensive chemical modification within cells through the activity of tRNA methyltransferase enzymes (TRMs). Although tRNA modifications are dynamic, how they impact cell behavior after stress and during tumorigenesis is not well understood. This review discusses how tRNA modifications influence the translation of codon-biased transcripts involved in responses to oxidative stress. We further discuss emerging mechanistic details about how aberrant TRM activity in cancer cells can direct programs of codon-biased translation that drive cancer cell phenotypes. The studies reviewed here predict future preventative therapies aimed at augmenting TRM activity in individuals at risk for cancer due to exposure. They further predict that attenuating TRM-dependent translation in cancer cells may limit disease progression while leaving noncancerous cells unharmed.

DNA damaging agents trigger the expression of the HML silent mating type locus in Saccharomyces cerevisiae.

Many DNA damaging agents also react with RNA and protein, and could thus cause epigenetic as well as genotoxic changes. To investigate which DNA damaging agents alter epigenetic states, we studied the chemical-induced changes in expression of the yeast silent mating type locus HMLα, which can be triggered by inhibiting yeast Sir2. We observed that the alkylating agent methyl methane sulfonate (MMS) can result in HMLα expression, using a colony sector assay that results from expression of a HML-positioned cre gene. Using single-cell imaging we also observed that alkylating agents, including MMS and methyl-3-nitro-1-nitrosoguanidine (MNNG), as well as short-wave UV, also decreased HML silencing. We suggest that chemical-induced alterations in heterochromatin structure could confer transient phenotypic changes that affect the cellular responses to DNA damaging agents.

Editor's Highlight: High-Throughput Functional Genomics Identifies Modulators of TCE Metabolite Genotoxicity and Candidate Susceptibility Genes.

Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcinogen. Reactive metabolites are implicated in renal carcinogenesis associated with TCE exposure, yet the toxicity mechanisms of these metabolites and their contribution to cancer and other adverse effects remain unclear. We employed an integrated functional genomics approach that combined functional profiling studies in yeast and avian DT40 cell models to provide new insights into the specific mechanisms contributing to toxicity associated with TCE metabolites. Genome-wide profiling studies in yeast identified the error-prone translesion synthesis (TLS) pathway as an import mechanism in response to TCE metabolites. The role of TLS DNA repair was further confirmed by functional profiling in DT40 avian cell lines, but also revealed that TLS and homologous recombination DNA repair likely play competing roles in cellular susceptibility to TCE metabolites in higher eukaryotes. These DNA repair pathways are highly conserved between yeast, DT40, and humans. We propose that in humans, mutagenic TLS is favored over homologous recombination repair in response to TCE metabolites. The results of these studies contribute to the body of evidence supporting a mutagenic mode of action for TCE-induced renal carcinogenesis mediated by reactive metabolites in humans. Our approach illustrates the potential for high-throughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.

Both RAD5-dependent and independent pathways are involved in DNA damage-associated sister chromatid exchange in budding yeast.

Sister chromatids are preferred substrates for recombinational repair after cells are exposed to DNA damage. While some agents directly cause double-strand breaks (DSBs), others form DNA base adducts which stall or impede the DNA replication fork. We asked which types of DNA damage can stimulate SCE in budding yeast mutants defective in template switch mechanisms and whether PCNA polyubiquitination functions are required for DNA damage-associated SCE after exposure to potent recombinagens. We measured spontaneous and DNA damage-associated unequal sister chromatid exchange (uSCE) in yeast strains containing two fragments of his3 after exposure to MMS, 4-NQO, UV, X rays, and HO endonuclease-induced DSBs. We determined whether other genes in the pathway for template switching, including UBC13, MMS2, SGS1, and SRS2 were required for DNA damage-associated SCE. RAD5 was required for DNA damage-associated SCE after exposure to UV, MMS, and 4-NQO, but not for spontaneous, X-ray-associated, or HO endonuclease-induced SCE. While UBC13, MMS2, and SGS1 were required for MMS and 4NQO-associated SCE, they were not required for UV-associated SCE. DNA damage-associated recombination between his3 recombination substrates on non-homologous recombination was enhanced in rad5 mutants. These results demonstrate that DNA damaging agents that cause DSBs stimulate SCE by RAD5-independent mechanisms, while several potent agents that generate bulky DNA adducts stimulate SCE by multiple RAD5-dependent mechanisms. We suggest that DSB-associated recombination that occurs in G2 is RAD5-independent.

An in vitro system for measuring genotoxicity mediated by human CYP3A4 in Saccharomyces cerevisiae.

P450 activity is required to metabolically activate many chemical carcinogens, rendering them highly genotoxic. CYP3A4 is the most abundantly expressed P450 enzyme in the liver, accounting for most drug metabolism and constituting 50% of all hepatic P450 activity. CYP3A4 is also expressed in extrahepatic tissues, including the intestine. However, the role of CYP3A4 in activating chemical carcinogens into potent genotoxins is unclear. To facilitate efforts to determine whether CYP3A4, per se, can activate carcinogens into potent genotoxins, we expressed human CYP3A4 in the DNA-repair mutant (rad4 rad51) strain of budding yeast Saccharomyces cerevisiae and tested the novel, recombinant yeast strain for ability to report CYP3A4-mediated genotoxicity of a well-known genotoxin, aflatoxin B1 (AFB1 ). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac, a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB1 , the expression of CYP3A4 supported DNA adduct formation, chromosome rearrangements, cell death, and expression of the large subunit of ribonucleotide reductase, Rnr3, a marker of DNA damage. Expression of CYP3A4 also conferred sensitivity in rad4 rad51 mutants exposed to colon carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx). These data confirm the ability of human CYP3A4 to mediate the genotoxicity of AFB1 , and illustrate the usefulness of the CYP3A4-expressing, DNA-repair mutant yeast strain for screening other chemical compounds that are CYP3A4 substrates, for potential genotoxicity. Environ. Mol. Mutagen. 58:217-227, 2017. © 2017 Wiley Periodicals, Inc.

CYP1A1 I462V polymorphism is associated with reduced genotoxicity in yeast despite positive association with increased cancer risk.

CYP1A1 functions in detoxifying xenobiotics but occasionally converts compounds into potent genotoxins. CYP1A1 activates polyaromatic hydrocarbons, such as benzo[a]pyrene 7,8 dihydrodiol (BaP-DHD), rendering them genotoxic. Particular alleles of CYP1A1, such as CYP1A1 I462V have been correlated with a higher incidence of breast and lung cancer, but it is unknown whether these variants express enzymes in vivo that are more potent in generating genotoxins. We individually expressed CYP1A1 (CYP1A1.1), CYP1A1 T461N (CYP1A1.4) and I462V (CYP1A1.2) alleles in wild-type and DNA repair deficient mutant strains of Saccharomyces cerevisiae (budding yeast) and asked which yeast strains exhibited the highest levels of carcinogen-associated genotoxicity after exposure to BaP-DHD, aflatoxin B1 (AFB1), and heterocyclic aromatic amines (HAAs). We measured carcinogen-associated recombination, Rad51 foci, and carcinogen-associated toxicity in a DNA repair mutant deficient in both nucleotide excision repair and recombinational repair. CYP1A1 activity was confirmed by measuring ethoxyresorufin-O-deethylation (EROD) activities. Our data indicate that CYP1A1 I462V allele confers the least carcinogen-associated genotoxicity, compared to CYP1A1; however, results vary depending on the chemical carcinogen and the genotoxic endpoint. We speculate that the cancer-associated risk of CYP1A1 I462V may be caused by exposure to other xenobiotics.

Nucleotide salvage deficiencies, DNA damage and neurodegeneration.

Nucleotide balance is critically important not only in replicating cells but also in quiescent cells. This is especially true in the nervous system, where there is a high demand for adenosine triphosphate (ATP) produced from mitochondria. Mitochondria are particularly prone to oxidative stress-associated DNA damage because nucleotide imbalance can lead to mitochondrial depletion due to low replication fidelity. Failure to maintain nucleotide balance due to genetic defects can result in infantile death; however there is great variability in clinical presentation for particular diseases. This review compares genetic diseases that result from defects in specific nucleotide salvage enzymes and a signaling kinase that activates nucleotide salvage after DNA damage exposure. These diseases include Lesch-Nyhan syndrome, mitochondrial depletion syndromes, and ataxia telangiectasia. Although treatment options are available to palliate symptoms of these diseases, there is no cure. The conclusions drawn from this review include the critical role of guanine nucleotides in preventing neurodegeneration, the limitations of animals as disease models, and the need to further understand nucleotide imbalances in treatment regimens. Such knowledge will hopefully guide future studies into clinical therapies for genetic diseases.

Activation of aflatoxin B1 by expression of human CYP1A2 polymorphisms in Saccharomyces cerevisiae.

Human susceptibility to environmental carcinogens is highly variable and depends on multiple genetic factors, including polymorphisms in cytochrome P450 genes. Although epidemiological studies have identified individual polymorphisms in cytochrome P450 genes that may alter cancer risk, there is often conflicting data about whether such polymorphisms alter the genotoxicity of environmental carcinogens. This is particularly true of the CYP1A2 polymorphisms that confer differential activation of multiple human carcinogens. To determine whether a single cytochrome P450 polymorphism confers higher levels of carcinogen-associated genotoxicity, we chose an organism that lack enzymes to metabolically activate aflatoxins and expressed individual human P450 genes in budding yeast. We measured the frequencies of recombination, Rad51 foci formation, 7-methoxyresorufin O-demethylase activities, and the concentrations of carcinogen-associated DNA adducts in DNA repair proficient yeast expressing P450 polymorphisms after exposure to aflatoxin B1 (AFB1).We measured growth of rad4 rad51 cells expressing CYP1A2 polymorphisms while exposed to AFB1. We observed that there was significantly less AFB1-associated genotoxicity in yeast expressing CYP1A2 I386F, while yeast expressing CYP1A2 C406Y exhibited intermediate levels of genotoxicity compared to yeast expressing CYP1A2 D348N or wild type. We conclude that differences in carcinogen genotoxicity can be observed in yeast expressing different CYP1A2 alleles. This is the first report that carcinogen-associated P450 polymorphisms can be studied in yeast.

Aflatoxin B(1)-Associated DNA Adducts Stall S Phase and Stimulate Rad51 foci in Saccharomyces cerevisiae.

AFB(1) is a potent recombinagen in budding yeast. AFB(1) exposure induces RAD51 expression and triggers Rad53 activation in yeast cells that express human CYP1A2. It was unknown, however, when and if Rad51 foci appear. Herein, we show that Rad53 activation correlates with cell-cycle delay in yeast and the subsequent formation of Rad51 foci. In contrast to cells exposed to X-rays, in which Rad51 foci appear exclusively in G2 cells, Rad51 foci in AFB(1)-exposed cells can appear as soon as cells enter S phase. Although rad51 and rad4 mutants are mildly sensitive to AFB(1), chronic exposure of the NER deficient rad4 cells to AFB(1) leads to increased lag times, while rad4 rad51 double mutants exhibit synergistic sensitivity and do not grow when exposed to 50 µM AFB(1). We suggest RAD51 functions to facilitate DNA replication after replication fork stalling or collapse in AFB(1)-exposed cells.

Elevated dNTP levels suppress hyper-recombination in Saccharomyces cerevisiae S-phase checkpoint mutants.

MEC1, the essential yeast homolog of the human ATR/ATM genes, controls the S-phase checkpoint and prevents replication fork collapse at slow zones of DNA replication. The viability of hypomorphic mec1-21 is reduced in the rad52 mutant, defective in homologous recombination, suggesting that replication generates recombinogenic lesions. We previously observed a 6-, 10- and 30-fold higher rate of spontaneous sister chromatid exchange (SCE), heteroallelic recombination and translocations, respectively, in mec1-21 mutants compared to wild-type. Here we report that the hyper-recombination phenotype correlates with lower deoxyribonucleoside triphosphate (dNTP) levels, compared to wild-type. By introducing a dun1 mutation, thus eliminating inducible expression of ribonucleotide reductase in mec1-21, rates of spontaneous SCE increased 15-fold above wild-type. All the hyper-recombination phenotypes were reduced by SML1 deletions, which increase dNTP levels. Measurements of dNTP pools indicated that, compared to wild-type, there was a significant decrease in dNTP levels in mec1-21, dun1 and mec1-21 dun1, while the dNTP levels of mec1-21 sml1, mec1-21 dun1 sml1 and sml1 mutants were approximately 2-fold higher. Interestingly, higher dNTP levels in mec1-21 dun1 sml1 correlate with approximately 2-fold higher rate of spontaneous mutagenesis, compared to mec1-21 dun1. We suggest that higher dNTP levels in specific checkpoint mutants suppress the formation of recombinogenic lesions.

Hypoxia enhances the replication of oncolytic herpes simplex virus in p53- breast cancer cells.

Hypoxic cancer cells are refractory to conventional chemotherapy. Herpes simplex virus type-1 (HSV-1)-derived oncolytic viruses are safe for therapy since they lack the neurovirulence gene ICP34.5. Cancer cells containing high MEK activity are permissive to the HSV-1-derived oncolytic virus, R3616. Considering that hypoxia increases MEK activity, we determined whether hypoxic MDA-MB-231 and MCF-7 cells were more permissive to R3616, compared to normoxic cells. We observed nine-fold higher (3.5 x 10e6 pfu/4 x 10e5 pfu/ml) titers in MDA-MB-231 hypoxic cells compared to normoxic; however, hypoxic MCF-7 cells did not yield higher R3616 titers. Markers for early and late viral infection were consistent with this result: (1) virus-induced chaperone-enriched (VICE) domains were observed in MDA-MB-231 cells, and (2) the HSV-1 glycoprotein C (gC), a protein produced late in infection, accumulated in hypoxic MDA-MB-231 cells. Thus, oncolytic R3616 virus may target hypoxic p53(-) breast cancer cells.

UV but not X rays stimulate homologous recombination between sister chromatids and homologs in a Saccharomyces cerevisiae mec1 (ATR) hypomorphic mutant.

MEC1, the essential yeast ATM/ATR homolog, prevents replication fork collapse and is required for the cellular response to DNA damage. We had previously observed higher rates of spontaneous SCE, heteroallelic recombination and translocations in mec1-21 mutants, which still retain some G2 checkpoint function, compared to mec1 null mutants, which are completely defective in checkpoint function, and wild type. However, the types of DNA lesions that are more recombinogenic in mec1-21, compared to wild type, are unknown. Here, we measured DNA damage-associated SCE, homolog (heteroallelic) recombination, and homology-directed translocations in mec1-21, and characterized types of DNA damage-associated chromosomal rearrangements that occur in mec1-21. Although frequencies of UV-associated recombination were higher in mec1-21, the mutant was defective in double-strand break-associated SCE and heteroallelic recombination. Over-expression of Rad53 in mec1-21 reduced UV-associated recombination but did not suppress the defect in X-ray-associated recombination. Both X ray and UV exposure increased translocation frequencies in mec1-21, but the majority of the UV-associated products were non-reciprocal translocations. We suggest that although recombinational repair of double-stand breaks is less efficient in mec1 mutants, recombinants may be generated by other mechanisms, such as break-induced replication.

The Saccharomyces cerevisiae checkpoint genes RAD9, CHK1 and PDS1 are required for elevated homologous recombination in a mec1 (ATR) hypomorphic mutant.

Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homologous recombination. The genetic requirements for hyper-recombination in ATR mutants are unknown. MEC1, the essential yeast ATR/ATM homolog, controls S and G(2) checkpoints and the DNA damage-inducibility of genes after radiation exposure. Since the mec1-Delta (null) mutant is defective in both S and G(2) checkpoints, we measured spontaneous and DNA damage-associated sister chromatid exchange (SCE), homolog (heteroallelic) recombination, and homology-directed translocations in the mec1-21 hypomorphic mutant, which is defective in the S phase checkpoint but retains some G(2) checkpoint function. We observed a sixfold, tenfold and 30-fold higher rate of spontaneous SCE, heteroallelic recombination, and translocations, respectively, in mec1-21 mutants compared to wild type. The mec1-21 hyper-recombination was partially reduced in rad9, pds1 and chk1 mutants, and abolished in rad52 mutants, suggesting the hyper-recombination results from RAD52-dependent recombination pathway(s) that require G(2) checkpoint functions. The HU and UV sensitivities of mec1-21 rad9 and mec1-21 rad52 were synergistically increased, compared to the single mutants, indicating that mec1-21, rad52 and rad9 mutants are defective in independent pathways for HU and UV resistance. G(2)-arrested mec1-21 rad9 cells exhibit more UV resistance than non-synchronized cells, indicating that one function of RAD9 in conferring UV resistance in mec1-21 is by triggering G(2) arrest. We suggest that checkpoint genes that function in the RAD9-mediated pathway are required for either homologous recombination or DNA damage resistance in the S phase checkpoint mutant mec1-21.

Stimulation of sister chromatid exchanges and mutation by aflatoxin B1-DNA adducts in Saccharomyces cerevisiae requires MEC1 (ATR), RAD53, and DUN1.

The hepatocarcinogen aflatoxin B(1) (AFB(1)) is a potent recombinagen but weak mutagen in the yeast Saccharomyces cerevisiae. AFB(1) exposure induces DNA damage-inducible genes, such as RAD51 and those encoding ribonucleotide reductase (RNR), through a MEC1 (ATR homolog)-dependent pathway. Previous studies have indicated that MEC1 is required for both AFB(1)-associated recombination and mutation, and suggested that AFB(1)-DNA adducts are common substrates for recombination and mutagenesis. However, little is known about the downstream effectors of MEC1 required for genotoxic events associated with AFB(1) exposure. Here we show that AFB(1) exposure increases frequencies of RAD51-dependent unequal sister chromatid exchange (SCE) and activates Rad53 (CHK2). We found that MEC1, RAD53, and DUN1 are required for both AFB(1)-associated mutation and SCE. Deletion of SML1, which encodes an inhibitor of RNR, did not suppress the DUN1-dependent requirement for AFB(1)-associated genetic events, indicating that higher dNTP levels could not suppress the dun1 phenotype. We identified AFB(1)-DNA adducts and show that approximately the same number of adducts are obtained in both wild type and rad53 mutants. Since DUN1 is not required for UV-associated mutation and recombination, these studies define a distinct role for DUN1 in AFB(1)-associated mutagenesis and recombination. We speculate that AFB(1)-associated DNA adducts stall DNA replication, a consequence of which can either be mutation or recombination.