CAN1 Gene Research Articles

The arginine transporter Can1 negatively regulates biofilm formation in yeasts.

The arginine transporter Can1 is a multifunctional protein of the conventional yeast Saccharomyces cerevisiae. Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the S. cerevisiae CAN1 gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the CAN1 knockout strain is rescued by a functional deficiency of the FLO8 gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the CAN1 knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the CAN1 knockout strain of the pathogenic yeast Candida glabrata exhibited slower growth and higher biofilm formation, similar to S. cerevisiae. More importantly, the C. glabrata CAN1 gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents.

A selection-based next generation sequencing approach to develop robust, genotype-specific mutation profiles in Saccharomyces cerevisiae.

Distinct mutation signatures arise from environmental exposures and/or from defects in metabolic pathways that promote genome stability. The presence of a particular mutation signature can therefore predict the underlying mechanism of mutagenesis. These insults to the genome often alter dNTP pools, which itself impacts replication fidelity. Therefore, the impact of altered dNTP pools should be considered when making mechanistic predictions based on mutation signatures. We developed a targeted deep-sequencing approach on the CAN1 gene in Saccharomyces cerevisiae to define information-rich mutational profiles associated with distinct rnr1 backgrounds. Mutations in the activity and selectivity sites of rnr1 lead to elevated and/or unbalanced dNTP levels, which compromises replication fidelity and increases mutation rates. The mutation spectra of rnr1Y285F and rnr1Y285A alleles were characterized previously; our analysis was consistent with this prior work but the sequencing depth achieved in our study allowed a significantly more robust and nuanced computational analysis of the variants observed, generating profiles that integrated information about mutation spectra, position effects, and sequence context. This approach revealed previously unidentified, genotype-specific mutation profiles in the presence of even modest changes in dNTP pools. Furthermore, we identified broader sequence contexts and nucleotide motifs that influenced variant profiles in different rnr1 backgrounds, which allowed specific mechanistic predictions about the impact of altered dNTP pools on replication fidelity.

Effect of the Deletion of Genes Related to Amino Acid Metabolism on the Production of Higher Alcohols by Saccharomyces cerevisiae.

The higher alcohols produced by Saccharomyces cerevisiae exert remarkable influence on the taste and flavour of Chinese Baijiu. In order to study the regulation mechanism of amino acid metabolism genes on higher alcohol production, eight recombinant strains with amino acid metabolism gene deletion were constructed. The growth, fermentation performance, higher alcohol production, and expression level of genes in recombinant and original α5 strains were determined. Results displayed that the total higher alcohol concentration in α5ΔGDH1 strain decreased by 27.31% to 348.68 mg/L compared with that of α5. The total content of higher alcohols in α5ΔCAN1 and α5ΔGAT1 strains increased by 211.44% and 28.36% to 1493.96 and 615.73 mg/L, respectively, compared with that of α5. This study is the first to report that the CAN1 and GAT1 genes have great influence on the generation of higher alcohols. The results demonstrated that amino acid metabolism plays a substantial role in the metabolism of higher alcohols by S. cerevisiae. Interestingly, we also found that gene knockout downregulated the expression levels of the knocked out gene and other genes in the recombinant strain and thus affected the formation of higher alcohols by S. cerevisiae. This study provides worthy insights for comprehending the metabolic mechanism of higher alcohols in S. cerevisiae for Baijiu fermentation.

Application of bamboo leaves extract to Chinese yellow rice wine brewing for ethyl carbamate regulation and its mitigation mechanism

Bamboo leaves extract (BLE) contains various effective ingredients, including phenolic compounds. In this study, the effect of BLE on ethyl carbamate (EC) formation was investigated in Chinese yellow rice wine brewing with three different fermentation starters (Saccharomyces cerevisiae, Saccharomyces cerevisiae and Lactobacillus brevis, and Chinese yeast). As a result, BLE showed significant inhibition effect on EC in multi-microbial fermented rice wine, by preventing the reactions between urea/citrulline and ethanol. We found that BLE had influence on arginine transport (GAP1, CAN1, ALP1, and VBA2 gene) in Saccharomyces cerevisiae (S. cerevisiae), which significantly up-regulated arginine uptake gene expression in vacuole (VBA2 gene) so that inhibited arginine metabolism. Besides, the presence of BLE could improve the overall quality of Chinese yellow rice wine. Consequently, it was worthwhile applying BLE to Chinese rice wine fermentation, especially the wine brewing with S. cerevisiae and Lactobacillus brevis starter.

An undergraduate laboratory module that uses the CRISPR/Cas9 system to generate frameshift mutations in yeast.

The CRISPR/Cas9 system is a powerful tool for gene editing and it has become increasingly important for biology students to understand this emerging technique. Most CRISPR laboratory teaching modules use complex metazoan systems or mammalian cell culture which can be expensive. Here, we present a lab module that engages students in learning the fundamentals of CRISPR/Cas9 methodology using the simple and inexpensive model system, Saccharomyces cerevisiae. Students use CRISPR/Cas9 and nonhomologous end joining to generate frameshift insertion and deletion mutations in the CAN1 gene, which are easily selected for using media plates that have canavanine. DNA sequencing is also performed to determine what type of mutation occurred in gene-edited cells. This easy to implement set of experiments has been run as both a 5-week and a shorter 3-week lab module. Learning assessments demonstrate increased understanding in CRISPR-related concepts as well as increased confidence using molecular techniques. Thus, this CRISPR/Cas9 lab module can be added to an existing Genetics, Microbiology, or Molecular Biology lab course to help undergraduate students learn current gene editing techniques with limited effort and cost. © 2019 International Union of Biochemistry and Molecular Biology, 47(5):573-580, 2019.

A Novel Tool for Microbial Genome Editing Using the Restriction-Modification System.

Scarless genetic manipulation of genomes is an essential tool for biological research. The restriction-modification (R-M) system is a defense system in bacteria that protects against invading genomes on the basis of its ability to distinguish foreign DNA from self DNA. Here, we designed an R-M system-mediated genome editing (RMGE) technique for scarless genetic manipulation in different microorganisms. For bacteria with Type IV REase, an RMGE technique using the inducible DNA methyltransferase gene, bceSIIM (RMGE-bceSIIM), as the counter-selection cassette was developed to edit the genome of Escherichia coli. For bacteria without Type IV REase, an RMGE technique based on a restriction endonuclease (RMGE-mcrA) was established in Bacillus subtilis. These techniques were successfully used for gene deletion and replacement with nearly 100% counter-selection efficiencies, which were higher and more stable compared to conventional methods. Furthermore, precise point mutation without limiting sites was achieved in E.coli using RMGE-bceSIIM to introduce a single base mutation of A128C into the rpsL gene. In addition, the RMGE-mcrA technique was applied to delete the CAN1 gene in Saccharomyces cerevisiae DAY414 with 100% counter-selection efficiency. The effectiveness of the RMGE technique in E.coli, B.subtilis, and S.cerevisiae suggests the potential universal usefulness of this technique for microbial genome manipulation.

CAN1 Arginine Permease Deficiency Extends Yeast Replicative Lifespan via Translational Activation of Stress Response Genes

Transcriptional regulation plays an important role in the control of gene expression during aging. However, translation efficiency likely plays an equally important role in determining protein abundance, but it has been relatively understudied in this context. Here, we used RNA sequencing (RNA-seq) and ribosome profiling to investigate the role of translational regulation in lifespan extension by CAN1 gene deletion in yeast. Through comparison of the transcriptional and translational changes in cells lacking CAN1 with other long-lived mutants, we were able to identify critical regulatory factors, including transcription factors and mRNA-binding proteins, that coordinate transcriptional and translational responses. Together, our data support a model in which deletion of CAN1 extends replicative lifespan through increased translation of proteins that facilitate cellular response to stress. This study extends our understanding of the importance of translational control in regulating stress resistance and longevity.

Growth conditions that increase or decrease lifespan in Saccharomyces cerevisiae lead to corresponding decreases or increases in rates of interstitial deletions and non-reciprocal translocations.

BackgroundAccumulation of DNA damage, mutations, and chromosomal abnormalities is associated with aging in many organisms. How directly various forms of genomic instability contribute to lifespan in different aging contexts is still under active investigation. Testing whether treatments that alter lifespan change mutation rates early during lifespan could provide support for genomic instability being at least partly responsible for changes in the rates of aging.ResultsRates of mutations, direct repeat recombination, or retrotransposition were measured in young cell populations from two strain backgrounds of Saccharomyces cerevisiae exposed to several growth conditions that shortened or extended yeast chronological lifespan. In most cases, rates of genomic instability did not consistently increase in young cells exposed to lifespan-shortening conditions or decrease in young cells exposed to lifespan-extending conditions. The mutation rate for a copy of the CAN1 gene integrated onto the right arm of chromosome VIII did show expected increases or decreases in young cells in the lifespan-altering growth conditions. These mutations were determined to frequently result from non-allelic recombination events, including non-reciprocal translocations, and were more strongly stimulated by using hydroxyurea to induce DNA replication stress than by the general DNA-damaging agent methyl methanesulfonate.ConclusionsThe results are not consistent with changes in mutation rates in general mediating the influence of alternative growth conditions on yeast lifespan. The strong correlation between non-allelic recombination events and the effects of the alternative growth conditions on lifespan indicates that genomic instability due to changes in recombination rates may directly contribute to the rate of aging or that lifespan-altering treatments may consistently increase or decrease DNA replication stress. These results further support the connection between DNA replication stress and aging observed in multiple organisms. Chromosomal abnormalities that likely arise from recombination events are more prevalent in multiple human tissues with increasing age, and further work in yeast could help to define mechanisms responsible for this observation and the impact of chromosomal abnormalities on aging.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-016-0447-5) contains supplementary material, which is available to authorized users.

Abstract 3580: Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements

Abstract Topoisomerase 2 (Top2) is the target of active anti-cancer agents such as etoposide and doxorubicin. These drugs interfere with the Top2 catalytic cycle and lead to trapping of the enzyme as a covalent protein: DNA complex. This trapped covalent complex is a unique DNA lesion that includes DNA strand breaks and covalent protein adducts at the site of the breaks. While Top2 mediated DNA damage is the major mechanism for tumor cell killing, it is also responsible for drug-induced translocations that can lead to secondary malignancies. We have developed several novel systems for assessing Top2-mediated genome instability using yeast as a model system. We developed repair proficient yeast strains that accumulate high levels of etoposide and other Top2 targeting drugs. We selected for loss-of-function mutations in the yeast CAN1 gene that arise following etoposide treatment, and examined the induced mutations by DNA sequencing. We identified a unique spectrum of mutations generated by etoposide in yeast. One novel class of mutational events induced by etoposide is relatively large deletions (>300 bp) and tandem duplications. Most of these etoposide-induced events are flanked by 4-8 nucleotide direct repeats. To further characterize the mechanism of Top2 induced genome instability, we took advantage of a novel Top2 allele isolated in our laboratory. This Top2 allele, termed top2AR, exhibits elevated levels of drug independent DNA cleavage in vitro, and inviability when combined with yeast mutants defective in DNA double strand break repair. We found a similar spectrum of large deletions and tandem duplications flanked by 4-8 nucleotide direct repeats. Interestingly, we also found that the recovery of large deletions and insertions was enhanced in yeast cells lacking Tdp1, a gene that participates in the repair of protein:DNA covalent complexes. Our results illustrate a novel type of genome rearrangement that is mediated by covalent Top2 DNA damage. It is noteworthy that the deletions and insertions flanked by short direct repeats have not been previously observed with other classes of DNA damaging agents. We suggest that the unique properties of these lesions may also contribute to oncogenic translocations induced by etoposide and other Top2 targeting drugs. We are currently using next-generation sequencing of etoposide treated yeast cells and cells carrying the top2AR allele to obtain a more complete picture of the types of alterations induced by Top2 mediated DNA damage. Citation Format: Matthew Gilbertson, Radhika Patel, Karin C. Nitiss, John L. Nitiss. Topoisomerase II mediated DNA damage generates unique classes of genome rearrangements. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3580.

Measuring UV-induced Mutagenesis at the CAN1 Locus in Saccharomyces cerevisiae

There are several methods to measure the capacity of yeast cell to respond to environmental impacts on their genome by mutating it. One frequently used method involves the detection of forward mutations in the CAN1 gene. The CAN1 gene encodes for an arginine permease that is responsible for the uptake of arginine and it can also transport the toxic analog of arginine, canavanine (Whelan et al., 1979). When CAN1 cells are grown on a media containing canavanine but lacking arginine, the cells die because of the uptake of the toxic canavanine. However, if a mutation in the CAN1 gene inactivates the permease, that cell survives and forms a colony on the plate. The following protocol describes the measurement of UV-induced mutagenesis at the CAN1 locus.

Exclusive induction of G:C to A:T transitions by 3-azido-1,2-propanediol in yeast

Sodium azide is a strong mutagen which has been successfully employed in mutation breeding of crop plants. In biological systems, it is metabolized to azidoalanine, but further bioactivation to a putative ultimate mutagen as well as the nature of the induced DNA modifications leading to mutations remain elusive. In this study, mutations induced in the CAN1 gene of yeast Saccharomyces cerevisiae by the representative mutagen 3-azido-1,2-propanediol (azidoglycerol, AZG) have been sequenced. Analysis of the forward mutation spectrum to canavanine resistance revealed that AZG induced nearly exclusively G:C to A:T transitions. AZG also induced reversions to tryptophan prototrophy by base-pair substitutions in a dose-dependent manner. This unusual mutational specificity may be shared by other organic azido compounds.

Abstract 2546: A novel DNA damage detection assay reveals a critical role of XPF in the repair of endogenous damage

Abstract Introduction: DNA damage, notably that resulting from defective nucleotide excision repair (NER), can lead to a number of diseases including those associated with neurodegeneration and cancer. XPF, a gene essential for effective NER, is typically associated with the repair of chemically and UV induced bulky DNA lesions as well as crosslinks. Recently, we developed a novel DNA damage detection assay (PADDA) capable of detecting at the nucleotide level, on a high-throughput scale, endogenous and induced DNA damage. We have previously validated this assay using multiple in vitro and in vivo approaches. By applying this assay to the study of wild-type (WT) and base excision repair (BER) defective cells, we have also reported the first direct evidence for preferential strand repair of endogenous DNA damage and documented the major role of BER in this process. In this study, we used PADDA to determine the role of XPF in the repair of endogenous DNA damage. Methods: Endogenous DNA damage was detected in the CAN1 gene by PADDA in wild-type and XPF defective S. cerevisiae cells in stationary phases of growth. Levels and locations of lesions were compared between strains. Locations of DNA damage were then compared to sites of previously reported mutations in the CAN1 gene using statistical approaches based on Chi-square goodness of fit and exact nonparametric tests. Results: PADDA revealed higher levels of endogenous DNA damage in XPF defective yeast cells compared to WT. Furthermore, a significantly higher rate of polymerase by-passable DNA damage was observed in XPF defective yeast cells compared to WT. There was a high association between the sites of by-passable endogenous DNA damage found in XPF defective cells to sites of previously reported mutations in the CAN1 gene. Conclusions: Our results establish yet another novel role for XPF protein: a major function in the repair of endogenous DNA damage. Furthermore, our results suggest a key XPF role in the repair of highly mutagenic by-passable DNA damage and therefore in the prevention of mutations caused by endogenous DNA damage. Given this important role, XPF may become a biomarker of cancer risk or even a suitable target for strategies aimed at preventing cancer and other diseases associated with DNA damage. Additionally, the ability of PADDA to map and quantify many different types of DNA damage suggests it may become a very important clinical tool to assess cancer risk and response to chemotherapy. Funding: This work was supported by the National Institute of Health [R03 CA117316-01] (AR) and the OUHSC Vice President for Research Fund (LQ). L.Q. holds a Presbyterian Health Foundation Endowed Chair in Otorhinolaryngology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2546. doi:1538-7445.AM2012-2546

Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases

BackgroundThe activity of degradative nucleases responsible for genomic DNA digestion has been observed in all kingdoms of life. It is believed that the main function of DNA degradation occurring during plant programmed cell death is redistribution of nucleic acid derived products such as nitrogen, phosphorus and nucleotide bases. Plant degradative nucleases that have been studied so far belong mainly to the S1-type family and were identified in cellular compartments containing nucleic acids or in the organelles where they are stored before final application. However, the explanation of how degraded DNA components are exported from the dying cells for further reutilization remains open.ResultsBioinformatic and experimental data presented in this paper indicate that two Arabidopsis staphylococcal-like nucleases, named CAN1 and CAN2, are anchored to the cell membrane via N-terminal myristoylation and palmitoylation modifications. Both proteins possess a unique hybrid structure in their catalytic domain consisting of staphylococcal nuclease-like and tRNA synthetase anticodon binding-like motifs. They are neutral, Ca2+-dependent nucleaces showing a different specificity toward the ssDNA, dsDNA and RNA substrates. A study of microarray experiments and endogenous nuclease activity revealed that expression of CAN1 gene correlates with different forms of programmed cell death, while the CAN2 gene is constitutively expressed.ConclusionsIn this paper we present evidence showing that two plant staphylococcal-like nucleases belong to a new, as yet unidentified class of eukaryotic nucleases, characterized by unique plasma membrane localization. The identification of this class of nucleases indicates that plant cells possess additional, so far uncharacterized, mechanisms responsible for DNA and RNA degradation. The potential functions of these nucleases in relation to their unique intracellular location are discussed.

Abstract C13: A novel DNA damage detection assay for prediction of individual cancer risk

Abstract Background: Unrepaired DNA damage leads to a host of diseases including cancer. Despite the profound impact of DNA damage on human health, little progress has been made in the development of assays capable of accurately measuring induced as well as endogenous in vivo DNA damage. We developed a novel primer-anchored DNA damage detection assay (PADDA) to map and quantify a broad range of DNA lesions at the nucleotide level. Our damage detection assay relies on the principle that the presence of DNA damage on the template can change the polymerase replicative rate, leading to lesion bypass with or without misincorporation, or to lesion-dependent replicative arrest before or opposite the damaged base. Methods: PADDA's specificity, sensitivity and dynamic range were first determined on artificial templates containing engineered DNA lesions and on hydrogen peroxide damaged DNA. To determine the sensitivity of PADDA to detect different spectra of in vivo DNA lesions, we mapped and quantified endogenous and induced DNA damage in wild-type (wt) and repair defective yeast and mice cells. In vivo spontaneous DNA lesions were mapped within the CAN1 gene of S. cerevisiae wt and base excision repair (BER) defective cells and compared to the location of CAN1 published mutations. In vivo induced DNA lesions were mapped within the TRP53 gene in wt and nucleotide excision repair mutant (Xpc−/−) mice irradiated with ultraviolet B (UVB) light. The location of nucleotide lesions were compared to the published p53 mutational spectrum for identical strains. Results: PADDA screens a specific DNA region for base damage, provides detailed fingerprint analysis of base damage and repair at the singlenucleotide level, and quantifies strand-specific DNA damage on a high-throughput scale. The sensitivity of PADDA to detect and map engineered lesions within synthetic oligonucleotides was 100% for abasic and 8-oxodA lesions, 85% for 8-oxoG lesions, and 67% for thymine glycol lesions. PADDA was also able to quantify in vitro induced DNA damage over a wide range of genotoxic exposures (1 μM to 500 mM hydrogen peroxide). Furthermore, PADDA detected significantly higher levels of endogenous damage in yeast cells in stationary phase than in exponential phase, and in yeast BER defective cells than in isogenic wt cells at any phase of growth. Importantly, PADDA detected levels of endogenous DNA lesions that are consistent with published estimates. When mapping endogenous DNA damage, several clusters of DNA lesions were identified in the CAN1 gene. Fisher's exact test showed that the damaged nucleotides detected by PADDA were associated with those previously reported to be the sites of mutations in CAN1. Similarly, a correlation was found between persistent UVB induced DNA damage in the non-transcribed strand of Xpc-/- mice and a previously reported mutational hotspot. These data demonstrate that PADDA is capable of identifying pre-mutagenic DNA lesions in vivo. Conclusion: PADDA is the first assay capable of accurately mapping and quantifying nucleotide and strand specific endogenous DNA damage. PADDA's unprecedented ability to map sites of persistent in vivo endogenous and induced DNA damage before mutation fixation and to quantify strand specific damage may become a critical tool for assessing cancer risk and prevention strategies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C13.

Targeted detection of in vivo endogenous DNA base damage reveals preferential base excision repair in the transcribed strand

Endogenous DNA damage is removed mainly via base excision repair (BER), however, whether there is preferential strand repair of endogenous DNA damage is still under intense debate. We developed a highly sensitive primer-anchored DNA damage detection assay (PADDA) to map and quantify in vivo endogenous DNA damage. Using PADDA, we documented significantly higher levels of endogenous damage in Saccharomyces cerevisiae cells in stationary phase than in exponential phase. We also documented that yeast BER-defective cells have significantly higher levels of endogenous DNA damage than isogenic wild-type cells at any phase of growth. PADDA provided detailed fingerprint analysis at the single-nucleotide level, documenting for the first time that persistent endogenous nucleotide damage in CAN1 co-localizes with previously reported spontaneous CAN1 mutations. To quickly and reliably quantify endogenous strand-specific DNA damage in the constitutively expressed CAN1 gene, we used PADDA on a real-time PCR setting. We demonstrate that wild-type cells repair endogenous damage preferentially on the CAN1 transcribed strand. In contrast, yeast BER-defective cells accumulate endogenous damage preferentially on the CAN1 transcribed strand. These data provide the first direct evidence for preferential strand repair of endogenous DNA damage and documents the major role of BER in this process.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.

DNA repair mutant pso2 of Saccharomyces cerevisiae is sensitive to intracellular acetaldehyde accumulated by disulfiram-mediated inhibition of acetaldehyde dehydrogenase

Blocking aldehyde dehydrogenase with the drug disulfiram leads to an accumulation of intracellular acetaldehyde, which negatively affects the viability of the yeast Saccharomyces cerevisiae. Mutants of the yeast gene PSO2, which encodes a protein specific for repair of DNA interstrand cross-links, showed higher sensitivity to disulfiram compared to the wild type. This leads us to suggest that accumulated acetaldehyde induces DNA lesions, including highly deleterious interstrand cross-links. Acetaldehyde induced the expression of a PSO2-lacZ reporter construct that is specifically inducible by bi- or poly-functional mutagens, e.g., nitrogen mustard and photo-activated psoralens. Chronic exposure of yeast cells to disulfiram and acute exposure to acetaldehyde induced forward mutagenesis in the yeast CAN1 gene. Disulfiram-induced mutability of a pso2Delta mutant was significantly increased over that of the isogenic wild type; however, this was not found for acetaldehyde-induced mutagenesis. Spontaneous mutability at the CAN1 locus was elevated in pso2Delta, suggesting that growth of glucose-repressed yeast produces DNA lesions that, in the absence of Pso2p-mediated crosslink repair, are partially removed by an error-prone DNA repair mechanism. The use of disulfiram in the control of human alcohol abuse increases cellular acetaldehyde pools, which, based on our observations, enhances the risk of mutagenesis and of other genetic damage.

DNA polymerase ζ is essential for hexavalent chromium-induced mutagenesis

Translesion synthesis (TLS) is a unique DNA damage tolerance mechanism involved in the replicative bypass of genetic lesions in favor of uninterrupted DNA replication. TLS is critical for the generation of mutations by many different chemical and physical agents, however, there is no information available regarding the role of TLS in carcinogenic metal-induced mutagenesis. Hexavalent chromium (Cr(VI))-containing compounds are highly complex genotoxins possessing both mutagenic and clastogenic activities. The focus of this work was to determine the impact that TLS has on Cr(VI)-induced mutagenesis in Saccharomyces cerevisiae. Wild-type yeast and strains deficient in TLS polymerases (i.e. Polζ ( rev3), Polη ( rad30)) were exposed to Cr(VI) and monitored for cell survival and forward mutagenesis at the CAN1 locus. In general, TLS deficiency had little impact on Cr(VI)-induced clonogenic lethality or cell growth. rad30 yeast exhibited higher levels of basal and induced mutagenesis compared to Wt and rev3 yeast. In contrast, rev3 yeast displayed attenuated Cr(VI)-induced mutagenesis. Moreover, deletion of REV3 in rad30 yeast ( rad30 rev3) resulted in a significant decrease in basal and Cr(VI) mutagenesis relative to Wt and rad30 single mutants indicating that mutagenesis primarily depended upon Polζ. Interestingly, rev3 yeast were similar to Wt yeast in susceptibility to Cr(VI)-induced frameshift mutations. Mutational analysis of the CAN1 gene revealed that Cr(VI)-induced base substitution mutations accounted for 83.9% and 100.0% of the total mutations in Wt and rev3 yeast, respectively. Insertions and deletions comprised 16.1% of the total mutations in Cr(VI) treated Wt yeast but were not observed rev3 yeast. This work provides novel information regarding the molecular mechanisms of Cr(VI)-induced mutagenesis and is the first report demonstrating a role for TLS in the fixation of mutations induced by a carcinogenic metal.

Identification of pathways controlling DNA damage induced mutation in Saccharomyces cerevisiae

Mutation in response to most types of DNA damage is thought to be mediated by the error-prone sub-branch of post-replication repair and the associated translesion synthesis polymerases. To further understand the mutagenic response to DNA damage, we screened a collection of 4848 haploid gene deletion strains of Saccharomyces cerevisiae for decreased damage-induced mutation of the CAN1 gene. Through extensive quantitative validation of the strains identified by the screen, we identified ten genes, which included error-prone post-replication repair genes known to be involved in induced mutation, as well as two additional genes, FYV6 and RNR4. We demonstrate that FYV6 and RNR4 are epistatic with respect to induced mutation, and that they function, at least partially, independently of post-replication repair. This pathway of induced mutation appears to be mediated by an increase in dNTP levels that facilitates lesion bypass by the replicative polymerase Polδ, and it is as important as error-prone post-replication repair in the case of UV- and MMS-induced mutation, but solely responsible for EMS-induced mutation. We show that Rnr4/Polδ-induced mutation is efficiently inhibited by hydroxyurea, a small molecule inhibitor of ribonucleotide reductase, suggesting that if similar pathways exist in human cells, intervention in some forms of mutation may be possible.

T2092 Distinct Cin and Min Pattern, CAN1 Gene Methylation Profile in African-Americans (AA) with Colon Cancer and Adenoma

T2092 Distinct Cin and Min Pattern, CAN1 Gene Methylation Profile in African-Americans (AA) with Colon Cancer and Adenoma