ABSTRACT: The cellular response to treatment with DNA-damaging substances at low concentrations which are genotoxic but do not have a strong cytotoxic effect are of special interest. In addition, environmental variations that influence growth conditions, e.g. different media, and individual fitness, e.g. different strains, are likely to influence and modulate the adverse effects of individual DNA damaging substances. At sub-cytotoxic levels, DNA damaging substances play an important role in the accumulation of genomic mutations. In longer living organisms, like humans and other mammals, exposure to DNA damaging substances over extended period of time is a critical factor that contributes to the development of various diseases and in particular of tumors. The aim of our work was to study how strain background and growth conditions influence respond to DNA damage caused by low doses of MMS and which part of these changes is responsible for their sensitivity to toxic conditions. We analyzed sensitivity of two yeast strains FF18984 and BY4742 to MMS in media with limited and full nutrient availability. Keywords: Yeast, S.cerevisiae, MMS, stress response, DNA damage Overall design: YPD and F1 media were inoculated with overnight pre-cultures and grown at 30°C to mid log-phase (OD600 0.6 to 0.8). Cultures were split into three parts: the first aliquot was mock-treated and used as control; the second and third aliquots were treated with low concentration of MMS (0.00125% and 0.0125%). All cultures were incubated at 30° C. Samples were collected after 30 min and 1h incubation. The total of 14 samples were analysed. With the FF18984 strain experiments were done in minimal (F1) and full (YPD) medium with both MMS concentrations (0.00125% and 0.0125%) for 30 min and 1h. With the BY4742 strain experiments were done in full (YPD) medium with 0.0125% MMS for 1h. As a reference we used mock-treated samples from the same medium and the same time point.
INSTRUMENT(S): Complementary DNA arrays produced with PCR fragments of 6116 ORFs of Saccharomyces cerevisiae
Project description:LSH/DDM1 enzymes are required for DNA methylation in higher eukaryotes and have poorly defined roles in genome maintenance in yeast, plants, and animals. The filamentous fungus Neurospora crassa is a tractable system that encodes a single LSH/DDM1 homolog (NCU06306). We report that the Neurospora LSH/DDM1 enzyme is encoded by mutagen sensitive-30 (mus-30), a locus identified in a genetic screen over 25 years ago. We show that MUS-30-deficient cells have normal DNA methylation, but are hypersensitive to the DNA damaging agent MMS (methyl methanesulfonate). MUS-30 is a nuclear protein, consistent with its predicted role as a chromatin remodeling enzyme, and levels of MUS-30 are increased following DNA damage. MUS-30 co-purifies with Neurospora WDR76, a homolog of yeast Changed Mutation Rate-1 and mammalian WD40 repeat domain 76. Deletion of wdr76 rescued MMS-hypersensitivity of Dmus-30 strains, demonstrating that the MUS-30-WDR76 interaction is functionally important. DNA damage-sensitivity of Dmus-30 is also partially suppressed by deletion of methyl adenine glycosylase-1, a component of the base excision repair machinery (BER); however, the rate of BER is not affected in Dmus-30 strains. It was reported that mammalian LSH is required for efficient double strand break (DSB) repair. We found that MUS-30-deficient cells were not defective for DSB repair, and we observed a negative genetic interaction between Dmus-30 and Dmei-3, the Neurospora RAD51 homolog required for homologous recombination. These data are consistent with a role for MUS-30 that is independent of DSB repair. Our findings demonstrate that LSH/DDM1 enzymes are key regulators of genome stability in eukaryotes. crf5-1 isolates (two replicates each from the F1 and F2 generation) were grown in Vogel's minimal medium for 48 hours. As a control, two replicates of the wildtype strain were grown under identical conditions.
Project description:DNA base damage is an important contributor to genome instability, but how the formation and repair of these lesions is affected by the genomic landscape is unknown. Here we describe genome-wide maps of DNA base damage, repair, and mutagenesis at single nucleotide resolution in yeast treated with the alkylating agent methyl methanesulfonate (MMS). Analysis of these maps revealed that base excision repair (BER) of alkylation damage is significantly modulated by chromatin, with faster repair in nucleosome free regions, and slower repair and higher mutation density within strongly positioned nucleosomes. Both the translational and rotational settings of lesions within nucleosomes significantly influence BER efficiency; moreover, this effect is asymmetric relative to the nucleosome dyad and is regulated by histone modifications. Our data also indicate that MMS-induced A mutations are significantly enriched on the non-transcribed strand (NTS) of yeast genes, particularly in BER-deficient strains, due to higher damage formation on the NTS and transcription-coupled repair of the transcribed strand (TS). These findings reveal the influence of chromatin on repair and mutagenesis of base lesions on a genome-wide scale, and suggest a novel mechanism for transcription-associated mutation asymmetry, which is frequently observed in human cancers. Overall design: MMS lesion mapping data was analyzed for WT or mag1∆ yeast cells treated with 0.2% MMS or 0.4% MMS and allowed to repair for 0hr, 30 minutes, 1 hour, or 2 hours.
Project description:Genomic instability is one of the hallmarks of cancer. Several chemotherapeutic drugs and radiotherapy induce DNA damage to prevent cancer cell replication. Cells in turn activate different DNA damage response (DDR) pathways to either repair the damage or induce cell death. These DDR pathways also elicit metabolic alterations which can play a significant role in the proper functioning of the cells. The understanding of these metabolic effects resulting from different types of DNA damage and repair mechanisms is currently lacking. In this study, we used NMR metabolomics to identify metabolic pathways which are altered in response to different DNA damaging agents. By comparing the metabolic responses in MCF-7 cells, we identified the activation of poly (ADP-ribose) polymerase (PARP) in methyl methanesulfonate (MMS)-induced DNA damage. PARP activation led to a significant depletion of NAD+. PARP inhibition using veliparib (ABT-888) was able to successfully restore the NAD+ levels in MMS-treated cells. In addition, double strand break induction by MMS and veliparib exhibited similar metabolic responses as zeocin, suggesting an application of metabolomics to classify the types of DNA damage responses. This prediction was validated by studying the metabolic responses elicited by radiation. Our findings indicate that cancer cell metabolic responses depend on the type of DNA damage responses and can also be used to classify the type of DNA damage.
Project description:Gene expression was compared for wild type yeast (BY4741) and yeast lacking Gal11/Med15 and Med3, or from a gal11-myc med3∆ strain. The gal11-myc allele shows a partial loss of function when combined with med3∆. Expression was analyzed for yeast grown in YPD as well as in CSM. We also examined gene expression of the wild type strain BY4742 grown in YPD and include that data here. Gene expression was compared for wild type yeast (BY4741 and BY4742) and yeast lacking Gal11/Med15 and Med3, or from a gal11-myc med3∆ strain.
Project description:Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1 Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Using regression correlation
Project description:Small Ubiquitin-like Modifiers play critical roles in the DNA Damage Response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment with the DNA damaging agent MMS. We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1 and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4.
Project description:Analysis of the response to hydroxyurea in a yeast yap1 mutant strain compared to wild-type strain (BY4741 or BY4742 backgrounds). Cells were grown in YPD rich medium containing 200 mM hydroxyurea (HU) for 2 hours.
Project description:Microarrays were conducted to asses the effect of Stb3 deletion in immediate transcriptional induction in response to glucose Keywords: Stb3, immediate glucose induction, growth genes. Overall design: Response of Stb3 delete cells was compared to that of wild type (BY4742) in cells grown for 3 days in YPD and cells that received 2% glucose for 10 minutes after the 3 day growth period. Two repetitive samples were done for each timepoint.
Project description:Analysis of the response to hydroxyurea in a yeast aft1 mutant strain compared to wild-type strain (BY4741 or BY4742 backgrounds). Cells were grown in YPD rich medium containong 200 mM hydroxyurea (HU) for 2 hours. Analysis of the response to hydroxyurea in a yeast aft1aft2 mutant strain (Y18aft2d) compared to wild-type strain (CM3260). Cells were grown in YPD rich medium containing 200 mM hydroxyurea (HU) for 2 hours. Gene expression changes due to the aft1 mutation were also analysed in absence of HU (BY4741 background). Analysis of mid-term response to hydroxyurea in a yeast wild-type strain (W303a). Cells were grown in YPD rich medium containing 200 mM hydroxyurea (HU) for 2 hours. Gene expression changes were analysed compared to the same strain grown in the same medium without HU. Keywords: repeat sample
Project description:Wild type and sgs1 null yeast were grown under DNA damaging (with MMS) conditions or without treatment to log phase and their transcriptional profiles compared. The human aging diseases Werner and Bloom syndromes are a result of mutation of the WRN and BLM genes, respectively. The SGS1 gene of Saccharomyces cerevisiae is homologous to the human WRN and BLM genes of the RecQ DNA helicase family. Deletion of SGS1 results in accelerated yeast aging and a reduction in life span as well as cell cycle arrest. We demonstrate that SGS1 deletion, DNA damage, and stress show similar transcriptional responses in yeast. Our comparative analysis of the genome-wide expression response of SGS1 deletion, stress and DNA damage indicates parallel transcriptional responses to cellular insult and aging in yeast.