Papers by Michael Fasullo
Genetics, Jul 1, 2001
Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated ... more Saccharomyces cerevisiae Rad51 is structurally similar to Escherichia coli RecA. We investigated the role of S. cerevisiae RAD51 in DNA damage-associated unequal sister chromatid exchanges (SCEs), translocations, and inversions. The frequency of these rearrangements was measured by monitoring mitotic recombination between two his3 fragments, his3-Delta5' and his3-Delta3'::HOcs, when positioned on different chromosomes or in tandem and oriented in direct or inverted orientation. Recombination was measured after cells were exposed to chemical agents and radiation and after HO endonuclease digestion at his3-Delta3'::HOcs. Wild-type and rad51 mutant strains showed no difference in the rate of spontaneous SCEs; however, the rate of spontaneous inversions was decreased threefold in the rad51 mutant. The rad51 null mutant was defective in DNA damage-associated SCE when cells were exposed to either radiation or chemical DNA-damaging agents or when HO endonuclease-induced double-strand breaks (DSBs) were directly targeted at his3-Delta3'::HOcs. The defect in DNA damage-associated SCEs in rad51 mutants correlated with an eightfold higher spontaneous level of directed translocations in diploid strains and with a higher level of radiation-associated translocations. We suggest that S. cerevisiae RAD51 facilitates genomic stability by reducing nonreciprocal translocations generated by RAD51-independent break-induced replication (BIR) mechanisms.
bioRxiv (Cold Spring Harbor Laboratory), May 7, 2019
Exposure to the mycotoxin aflatoxin B1 (AFB 1) strongly correlates with hepatocellular carcinoma.... more Exposure to the mycotoxin aflatoxin B1 (AFB 1) strongly correlates with hepatocellular carcinoma. P450 enzymes convert AFB 1 into a highly reactive epoxide that forms unstable 8,9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 (AFB 1-N 7-Gua) DNA adducts, which convert to stable mutagenic AFB 1 formamidopyrimidine (FAPY) DNA adducts. In CYP1A2-expressing budding yeast, AFB 1 is a weak mutagen but a potent recombinagen. However, few genes have been identified that confer AFB 1 resistance. Here, we profiled the yeast genome for AFB 1 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 M AFB 1 for 20 hs. By using next generation sequencing to count molecular barcodes, we identified 85 AFB 1 resistant genes from the CYP1A2-expressing libraries. While functionally diverse genes, including those that function in proteolysis, actin reorganization, and tRNA modification, were identified, those that function in post-replication DNA repair and encode proteins that bind to DNA damage were over-represented, compared to the yeast genome, at large. DNA metabolism genes included those functioning in DNA damage tolerance, checkpoint recovery and replication fork maintenance, emphasizing the potency of the mycotoxin to trigger replication stress. Among genes involved in error-free DNA damage tolerance, we observed that CSM2, a member of the CSM2(SHU) complex, functioned in AFB 1-associated sister chromatid recombination while suppressing AFB 1-associated mutations. These studies thus broaden the number of AFB 1 resistant genes and have elucidated a mechanism of error-free bypass of AFB 1associated DNA adducts.
Molecular Biology of the Cell, Sep 1, 2004
The potent carcinogen aflatoxin B 1 is a weak mutagen but a strong recombinagen in Saccharomyces ... more The potent carcinogen aflatoxin B 1 is a weak mutagen but a strong recombinagen in Saccharomyces cerevisiae. Aflatoxin B 1 exposure greatly increases frequencies of both heteroallelic recombination and chromosomal translocations. We analyzed the gene expression pattern of diploid cells exposed to aflatoxin B 1 using high-density oligonucleotide arrays comprising specific probes for all 6218 open reading frames. Among 183 responsive genes, 46 are involved in either DNA repair or in control of cell growth and division. Inducible growth control genes include those in the TOR signaling pathway and SPO12, whereas PKC1 is downregulated. Eleven of the 15 inducible DNA repair genes, including RAD51, participate in recombination. Survival and translocation frequencies are reduced in the rad51 diploid after aflatoxin B 1 exposure. In mec1 checkpoint mutants, aflatoxin B 1 exposure does not induce RAD51 expression or increase translocation frequencies; however, when RAD51 is constitutively overexpressed in the mec1 mutant, aflatoxin B 1 exposure increased translocation frequencies. Thus the transcriptional profile after aflatoxin B 1 exposure may elucidate the genotoxic properties of aflatoxin B 1 .
Copyright information: Taken from "Inverted repeat-stimulated sister-chromatid exchange even... more Copyright information: Taken from "Inverted repeat-stimulated sister-chromatid exchange events are -independent but reduced in a mutant"Nucleic Acids Research 2005;33(16):5243-5249.Published online 15 Sep 2005PMCID:PMC1216339.© The Author 2005. Published by Oxford University Press. All rights reserved () Pausing at the replication fork can lead to template switching. Template switching can form a wild-type gene. The large loop must be preserved from mismatch repair until the next round of DNA replication, or else be repaired in favor of loop retention, to generate a His cell. (). A DSB formed due to nicking of the secondary structure within the triplet repeat sequence may be repaired by an intra-chromosomal single-strand annealing-like event, leading to contraction of the repeat tract. The hatch marks represent the repeated units.
Journal of Clinical Toxicology, Nov 5, 2014
![Research paper thumbnail of High-Throughput Screening of the<i>Saccharomyces cerevisiae</i>Genome for 2-Amino-3-Methylimidazo [4,5-<i>f</i>] Quinoline Resistance Identifies Colon Cancer-Associated Genes](https://attachments.academia-assets.com/109252045/thumbnails/1.jpg)
bioRxiv (Cold Spring Harbor Laboratory), Oct 27, 2022
Heterocyclic aromatic amines (HAAs) are potent carcinogenic agents found in charred meats and cig... more 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-3methylimidazo[4,5-f]quinoline (IQ). CYP1A2 and NAT2 activate IQ to become a mutagenic nitrenium compound. We introduced an expression vector that contains human CYP1A2 and NAT2 genes into selected mutant strains and the diploid yeast deletion collection. The deletion libraries expressing CYP1A2 and NAT2 or no human genes were exposed to either 400 or 800 µM IQ for five or ten generations. DNA barcodes were sequenced using the Illumina HiSeq 2500 platform and statistical significance was determined for exactly matched barcodes. Four screens for IQ resistance in the "humanized" collection identified 1160 unique ORFs, of which 337 were validated or duplicated in at least two screens. Two screens of the original yeast library identified 101 genes that overlap with the 337 previously identified. Selected genes were validated by growth curves, competitive growth assays, or trypan blue assays. Prominent among both sets are ribosomal protein genes, while nitrogen metabolism, cell wall synthesis, and phosphatase genes were identified among the "humanized" library. Protein complexes identified included the casein kinase 2 (CK2) and histone chaperone (HIR) complex. DNA repair genes included NTG1, RAD18, RAD9, PSY2 and UBC13. Polymorphisms in human NTHL1, the NTG1 ortholog, and RAD18 are risk factors for colon cancer. These studies thus provoke questions of whether genetic risk factors for colon cancer confer more HAA-associated toxicity. .
Mutation Research, Nov 1, 2018
Many DNA damaging agents also react with RNA and protein, and could thus cause epigenetic as well... more 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 shortwave 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.
Toxicological Sciences, Aug 21, 2017
Trichloroethylene (TCE), an industrial chemical and environmental contaminant, is a human carcino... more 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 highthroughput in vitro functional profiling in yeast to elucidate toxicity pathways (molecular initiating events, key events) and candidate susceptibility genes for focused study.
PubMed, Dec 1, 1996
The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutageni... more The potent liver carcinogen aflatoxin B1 (AFB1) is metabolized by cytochrome P450 to the mutagenic epoxide. We have observed that activated AFB1 also strongly induced mitotic recombination in the yeast Saccharomyces cerevisiae. To compare the recombinogenicity of AFB1 to its mutagenicity, three metabolically competent S. cerevisiae strains have been constructed. The frequencies of induced recombinants resulting from gene conversion or chromosomal translocations were determined by different prototrophic selections using two strains, whereas the inducibility of forward mutations was determined by the frequency of drug resistance in the third strain. Human cytochrome P4501A1- (CYP1A) and NADPH-cytochrome P450-oxidoreductase cDNAs were expressed in the strains to ensure intracellular metabolism to the epoxide. Exposure of the strains to AFB1 resulted in a 139- and 24-fold increase in the translocation and gene conversion frequencies, respectively, whereas the mutation frequency was increased only 3-fold. In contrast, benzo[a]pyrene-7,8-dihydrodiol and ethyl methanesulfonate induced mutation and mitotic recombination to similar degrees. We conclude that AFB1 exerted a strong recombinogenic, but only a weak mutagenic, effect. The recombinogenicity of AFB1 in yeast may indicate a mechanism for the high proportion of loss of heterozygosity that has been detected in AFB1-related human liver cancers.
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Oct 15, 2005
DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous en... more DNA double-strand break (DSB) repair occurs by homologous recombination (HR) or non-homologous endjoining (NHEJ). In Saccharomyces cerevisiae, expression of both MATa and MATalpha inhibits NHEJ and facilitates DSB-initiated HR. We previously observed that DSB-initiated recombination between two his3 fragments, his3-Delta5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and his3-Delta3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;::HOcs is enhanced in haploids and diploids expressing both MATa and MATalpha genes, regardless of the position or orientation of the his3 fragments. Herein, we measured frequencies of DNA damage-associated translocations and sister chromatid exchanges (SCEs) in yku70 haploid mutants, defective in NHEJ. Translocation and SCE frequencies were measured in strains containing the same his3 fragments after DSBs were made directly at trp1::his3-Delta3&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;::HOcs. Wild type and yku70 cells were also exposed to ionizing radiation and radiomimetic agents methyl methanesulfonate (MMS), phleomycin, and 4-nitroquinolone-1-oxide (4-NQO). Frequencies of X-ray-associated and DSB-initiated translocations were five-fold higher in yku70 mutants compared to wild type; however, frequencies of phleomycin-associated translocations were lower in the yku70 haploid mutant. Frequencies of DSB-initiated SCEs were 1.8-fold higher in the yku70 mutant, compared to wild type. Thus, DSB-initiated HR between repeated sequences on non-homologous chromosomes and sister chromatids occurs at higher frequencies in yku70 haploid mutants; however, higher frequencies of DNA damage-associated HR in yku70 mutants depend on the DNA damaging agent.
Mutation Research: Fundamental And Molecular Mechanisms Of Mutagenesis, Mar 22, 2004
Saccharomyces cerevisiae rad9 checkpoint mutants exhibit pleiotropic phenotypes, including higher... more Saccharomyces cerevisiae rad9 checkpoint mutants exhibit pleiotropic phenotypes, including higher frequencies of chromosome loss, radiation sensitivity, and decreased induction of DNA damage-inducible genes. We had previously shown that rad9 mutants exhibit higher frequencies of DNA damage-associated translocations but lower frequencies of DNA damage-associated sister chromatid exchange (SCE), compared to wild type. Herein, we have shown that differences between the frequencies of DNA damage-associated recombination in the rad9 mutant and wild type depend on the identity and the concentration of the DNA damaging agent. Translocation and SCE frequencies were measured in strains containing truncated his3 fragments, located either on chromosomes II and IV, or located in tandem on chromosome IV, respectively. DNA damage-associated frequencies of translocations after exposure to hydrogen peroxide (H(2)O(2)), bleomycin, phleomycin, cisplatin, and camptothecin are higher in the rad9 diploid than in wild type. However, translocation frequencies after exposure to 4-nitroquinoline 1-oxide (4-NQO) and N-methyl-N&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-nitro-N-nitrosoguanidine (MNNG) are similar in rad9 and wild-type strains. We suggest that the deficiency in triggering G(2) arrest after exposure to specific DNA damaging agents results in the higher levels of DNA damage-associated translocations in rad9 mutants.
Cell Cycle, Aug 1, 2008
Specific ataxia telangiectasia and Rad3-related (ATR) mutations confer higher frequencies of homo... more 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-Δ (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.
Environmental and Molecular Mutagenesis, Apr 24, 2017
P450 activity is required to metabolically activate many chemical carcinogens, rendering them hig... more 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 (AFB 1). Yeast microsomes containing human CYP3A4, but not those that do not contain CYP3A4, were active in hydroxylation of diclofenac (DCF), a known CYP3A4 substrate drug, a result confirming CYP3A4 activity in the recombinant yeast strain. In cells exposed to AFB 1 , 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 AFB 1 , 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.
Molecular and Cellular Biology, Oct 1, 1988
We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (tr... more We used the his3 recombinational substrates (his3 fragments) to direct large interchromosomal (translocations) and intrachromosomal (deletions and tandem duplications) rearrangements in the yeast Saccharomyces cerevisiae. In strains completely deleted for the wild-type HIS3 gene, his3 fragments, one containing a deletion of 5' amino acid coding sequences and the other containing a deletion of 3' amino acid coding sequences, were first placed at preselected sites by homologous recombination. His' revertants that arose via spontaneous mitotic recombination between the two his3 fragments were selected. This strategy was used to direct rearrangements in both RAD52' and rad52 mutant strains. Translocations occurred in the RAD52+ genetic background and were characterized by orthogonal field alternating gel electrophoresis of yeast chromosomal DNA and by standard genetic techniques. An unexpected translocation was also identified in which HIS3 sequences were amplified. Two types of tandem duplications of the GAL(7, 10, 1) locus were also directed, and one type was not observed in rad52 mutants. Recombination mechanisms are discussed to account for these differences.
Nucleic Acids Research, May 15, 2003
Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those di... more Sister chromatid exchange (SCE) can occur by several recombination mechanisms, including those directly initiated by double-strand breaks (DSBs), such as gap repair and break-induced replication (BIR), and those initiated when DNA polymerases stall, such as template switching. To elucidate SCE recombination mechanisms, we determined whether spontaneous and DNA damage-associated SCE requires speci®c genes within the RAD52 and RAD3 epistasis groups in Saccharomyces cerevisiae strains containing two his3 fragments, his3-D5 ¢ and his3-D3 ¢::HOcs. SCE frequencies were measured after cells were exposed to UV, X-rays, 4-nitroquinoline 1-oxide (4-NQO) and methyl methanesulfonate (MMS), or when an HO endonuclease-induced DSB was introduced at his3-D3¢::HOcs. Our data indicate that genes involved in gap repair, such as RAD55, RAD57 and RAD54, are required for DNA damageassociated SCE but not for spontaneous SCE. RAD50 and RAD59, genes required for BIR, are required for X-ray-associated SCE but not for SCE stimulated by HO-induced DSBs. In comparison with wild type, rates of spontaneous SCE are 10-fold lower in rad51 rad1 but not in either rad51 rad50 or rad51 rad59 double mutants. We propose that gap repair mechanisms are important in DNA damageassociated recombination, whereas alternative pathways, including a template switch pathway, play a role in spontaneous SCE.
AIMS genetics, Aug 1, 2017
Sister chromatids are preferred substrates for recombinational repair after cells are exposed to ... more 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.
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Papers by Michael Fasullo