- Research article
- Open Access
The DNA-damage signature in Saccharomyces cerevisiae is associated with single-strand breaks in DNA
- Rebecca C Fry†1, 2,
- Michael S DeMott†1,
- Joseph P Cosgrove1, 3,
- Thomas J Begley4,
- Leona D Samson1, 2 and
- Peter C Dedon1, 2Email author
© Fry et al; licensee BioMed Central Ltd. 2006
Received: 04 August 2006
Accepted: 12 December 2006
Published: 12 December 2006
Upon exposure to agents that damage DNA, Saccharomyces cerevisiae undergo widespread reprogramming of gene expression. Such a vast response may be due not only to damage to DNA but also damage to proteins, RNA, and lipids. Here the transcriptional response of S. cerevisiae specifically induced by DNA damage was discerned by exposing S. cerevisiae to a panel of three "radiomimetic" enediyne antibiotics (calicheamicin γ1I, esperamicin A1 and neocarzinostatin) that bind specifically to DNA and generate varying proportions of single- and double-strand DNA breaks. The genome-wide responses were compared to those induced by the non-selective oxidant γ-radiation.
Given well-controlled exposures that resulted in similar and minimal cell death (~20–25%) across all conditions, the extent of gene expression modulation was markedly different depending on treatment with the enediynes or γ-radiation. Exposure to γ-radiation resulted in more extensive transcriptional changes classified both by the number of genes modulated and the magnitude of change. Common biological responses were identified between the enediynes and γ-radiation, with the induction of DNA repair and stress response genes, and the repression of ribosomal biogenesis genes. Despite these common responses, a fraction of the response induced by gamma radiation was repressed by the enediynes and vise versa, suggesting that the enediyne response is not entirely "radiomimetic." Regression analysis identified 55 transcripts with gene expression induction associated both with double- or single-strand break formation. The S. cerevisiae "DNA damage signature" genes as defined by Gasch et al.  were enriched among regulated transcripts associated with single-strand breaks, while genes involved in cell cycle regulation were associated with double-strand breaks.
Dissection of the transcriptional response in yeast that is specifically signaled by DNA strand breaks has identified that single-strand breaks provide the signal for activation of transcripts encoding proteins involved in the DNA damage signature in S. cerevisiae, and double-strand breaks signal changes in cell cycle regulation genes.
Exposure to DNA damaging agents can cause mutation and cell death and may ultimately lead to disease. Protection from this damage is provided by a host of DNA repair and cell cycle checkpoint proteins that collectively represent numerous pathways to help in the recovery response . In humans, there are approximately 150 DNA repair and cell cycle checkpoint proteins that serve to ensure the repair of damage caused to DNA [3, 4] and most of these have functional homologues in S. cerevisiae. Recently, studies have shown that proteins with additional cellular functions beyond DNA repair and cell cycle regulation may ameliorate the toxic effects of agents that cause DNA damage [5–8].
Genome-wide phenotyping studies to identify genes involved in cellular recovery after exposure to DNA alkylating agents, such as methyl methane sulfonate (MMS), indicate that a vast array of cellular processes are required for the recovery of S. cerevisiae. Transcriptional profiling demonstrated that up to 30% of the S. cerevisiae ~6000 genes respond upon exposure to MMS in a time-, agent- and dose-dependent manner [9, 10]. Given this vast transcriptional response to damaging agents like MMS and the knowledge that, in addition to DNA, these agents can also damage proteins, RNA and lipids, we set out to identify the transcriptional response specifically caused by damage to DNA.
Proportions of Single- and Double-Strand Damage Produced by γ-Radiation and Enediynes
Ratio of direct single- to double-strand breaks
Ratio of single- to double-strand deoxyribose oxidation events
Expression profiling experiments have been performed using some of these agents independently [13, 14]. However, there are no studies that directly compare the responses of these enediynes to each other and to that of γ-radiation under identical conditions. We establish four key findings: (i) under conditions of similar cell survival, exposure to non-selective γ-radiation results in more extensive reprogramming of S. cerevisiae transcription than does exposure to the DNA-selective enediynes; (ii) in response to DNA-strand breaks induced by both the non-selective and selective treatments, S. cerevisiae induces genes involved in DNA repair and the general stress response and represses genes encoding for ribosomal biogenesis; (iii) a considerable fraction of the response upon exposure to γ-radiation was not mimicked by treatment with the enediynes; and (iv) the "DNA damage signature" as described by Gasch et al.  in S. cerevisiae is associated with single-strand breaks in DNA. These results have implications for our understanding of the cellular responses arising from damage to specific components of the cell.
Results and discussion
Genome-wide responses of S. cerevisiae upon exposure to enediynes and γ-radiation are partially overlapping
Gene expression changes in response to enediyne or γ-radiation treatmenta
# of unique ORFS with ≥ 1.5-fold changeb
Calicheamicin γ 1 I
Common and unique responses of S. cerevisiae upon exposure to enediynes or γ-radiation
Common responses were identified to treatment with both enediynes and γ-radiation representing 55% of the modulated transcripts (124 of 225 ORFs). Genes induced by both enediynes and γ-radiation (Sector I) were represented by 13 Gene Ontology categories, 7 of which are involved in DNA repair [see Additional file 1]. The most significant class enriched was that of Response to Stress (p < 3.7 × 10-5) and includes MSN2, MSN4, DUN1, and RAD51. Genes repressed by both enediynes and γ-radiation (Sector III) were represented by 31 Gene Ontology categories. Among these 31 categories, significant enrichment was seen in the Ribosomal Large Subunit Biogenesis (p < 0.006) [see Additional file 1]. Interestingly, both the induction of stress response genes and repression of ribosomal protein synthesis genes are two of the hallmarks of the Environmental Stress Response (ESR) in S. cerevisiae. The ESR was identified by Brown and colleagues where a commonly responding set of genes (~900) was modulated upon exposure to a wide variety of environmental stressors including temperature shock, hydrogen peroxide and hyper- and hypo-osmotic shock .
Despite the common response to treatment with enediynes and γ-radiation described above, a substantial fraction (45%) of the genomic response induced by γ-radiation was repressed by the enediynes and vise versa suggesting that much of the response to the enediynes is not "radiomimetic." Specifically 101 transcripts of the total 225 modulated transcripts demonstrated opposite directionality between γ-radiation and the enediynes. Genes that were induced by γ-radiation but repressed by enediynes (Sector II) were enriched for 19 Gene Ontology categories [see Additional file 1]. The most significantly enriched class was Mutagenesis (p < 2 × 10-6) and includes POL30 and CDC8. The final class included transcripts repressed upon exposure to γ-radiation and induced by enediynes (Sector IV). A significantly enriched Gene Ontology class includes Chromatin Assembly/Disassembly (p < 0.0007) including HTB2, HHT1, and HHT2 [see Additional file 1].
Regression analysis identified gene induction that associates with double-strand or single-strand damage
Elevated expression of ORFs associated with single- and double-strand damage caused by exposure of S. cerevisiae to γ-radiation and enediynesa
Elevated Expression of ORFS Associated with Single-Strand Damage
Elevated Expression of ORFS Associated with Double-Strand Damage
YLR164Wp is homologous to TIM18p
Non-essential protein required for construction of the outer spore wall layers
adenine aminohydrolase (adenine deaminase)
Involved in daughter cell separation and Chromosome STability
The authentic, non-tagged protein was localized to the mitochondria
branched-chain amino acid transaminase
Protein required for cell viability
Core interacting component 1
alkyl hydroperoxide reductase
ATP dependent RNA helicase|dead box protein
chromatin assembly factor-I (CAF-I) p60 subunit
Cytoplasmic DExD/H-box helicase
Cdc28p kinase inhibitor
p24 protein involved in membrane trafficking
histone H3 (HHT1 and HHT2 code for identical proteins)
karyopherin beta 4
heat shock protein 26
ABC transporter (putative)|highly similar to Pdr5p
Mps One Binder
DNA polymerase I alpha subunit p180
Protein that localizes primarily to the plasma membrane
Rad51p colocalizes to ~65 spots with Dmc1p prior to synapsis
29% identical to the human p34 subunit of RF-A|replication factor RF-A subunit 2
ribonucleotide reductase subunit
Severe Depolymerization of Actin
ribosomal protein L28 (L29) (rp44) (YL24)
invertase (sucrose hydrolyzing enzyme)
ribosomal protein S25A (S31A) (rp45) (YS23)
similar to human translation initiation factor 6 (eIF6);
N2,N2-dimethylguanosine-specific tRNA methyltransferase
U3 snoRNP protein
Putative mitochondrial inner membrane transporter
Interestingly, ORFs with regulatory association with single-strand breaks show an enrichment of gene ontology categories including Response to Stress (HSP26, YCL033C, DUN1, RAD51, HYR1, ASF1, AHP1, CRS5), and DNA replication (POL1, RFA2) [see Additional file 2]. Within this single-strand break associated gene set there is an enrichment of genes belonging to the "DNA damage signature" in S. cerevisiae including DUN1, RAD51, RNR2, and YBR070C. The DNA damage signature, identified by Brown and colleagues, includes nine genes: the DNA damage repair genes RAD51 and RAD54, ribonucleotide reductase subunits RNR2 and RNR4, DNA damage activated kinase DUN1, and uncharacterized genes YER004W and YBR070C . The "DNA damage" signature was identified by comparing expression programs in S. cerevisiae that were modulated by a range of environmental stresses including heat shock, oxidative stress, reductive stress, osmotic shock and amino acid starvation to those elicited specifically by DNA damage induced by the alkylating agent methylmethane sulfonate (MMS) and ionizing radiation (γ-radiation) [1, 15]. Although it is not surprising that here we identify the DNA damage signature gene set as associated with γ-radiation (as this gene set was characterized in response to γ-radiation), it is noteworthy that there is a specificity of the activation of the DNA damage signature gene set to single-strand damage.
Comparisons with published studies of enediyne-induced changes in gene expression
Expression profiling experiments have been performed using calicheamicin  and neocarzinostatin . Several major experimental differences make it impossible to directly compare the data generated here with those of the published studies. The calicheamicin studies performed by Schaus et al. employed the YPH500α strain of S. cerevisiae and significantly higher (8–80 nM) drug concentrations , while the neocarzinostatin studies of Watanabe et al.  utilized strain BY4741 and the holoantibiotic form of the drug (DNA-cleaving chromophore bound to 17,000 Da apo-protein; see ref. ). In addition to the absence of publicly available expression data sets for these studies, neither reported any measure of enediyne-induced cytotoxicity or other biological response that could be used to normalize the resulting transcriptional data.
Genome-wide expression profiling in S. cerevisiae has resulted in the understanding that a robust response is mounted upon exposure to agents that damage DNA. Here we discern the components of the transcriptional response of S. cerevisiae that are specifically due to particular types of DNA damage, namely that of single- or double-strand DNA damage arising from deoxyribose oxidation. These studies were conducted with a panel of three DNA-selective enediyne antibiotics (calicheamicin γ1I, esperamicin A1 and neocarzinostatin) that produce different proportions of double- and single-strand deoxyribose damage in DNA, with genome-wide responses compared to those induced by the non-selective γ-radiation. We find at doses producing similar toxicity, exposure to non-selective γ-radiation results in more extensive reprogramming of the S. cerevisiae transcriptome than exposure to each of the three enediynes. The extensive response to γ-radiation may reflect the non-specific nature of the oxidative attack on DNA (base and sugar), lipids, carbohydrates, proteins and small metabolites in cells. A striking finding is that only a modest fraction of the response upon exposure to γ-radiation was mimicked by treatment with the enediynes. We find that, in response to DNA strand breaks induced by both the non-selective and selective agents, yeast induce genes involved in DNA repair and stress response and repress genes encoding for ribosomal biogenesis. We also identify that the DNA damage signature in S. cerevisiae is more closely associated with single-strand breaks in DNA, than with double-strand breaks.
Yeast strains, culture, and reagents
Saccharomyces cerevisiae strain DBY747 (MAT a, his3 Δ1, leu2 Δ112, ura3 Δ52, trp1 Δ289a gals, Can1, CUPr) was used in this study. Enediynes were used as methanolic stocks and obtained as follows: Calicheamicin was obtained from Wyeth Research (Cambridge, MA), neocarzinostatin was obtained from Kayaku Co. Ltd. (Tokyo, Japan; no longer available; currently produced by Sigma Chemical Company, St. Louis, MO), esperamicin A1 was obtained from Bristol Myers Squibb (Wallingford, CT; no longer available). The neocarzinostatin chromophore was isolated by methanol extraction and the concentration determined as described elsewhere . Cells were grown and maintained in YPD (10 g yeast extract, 20 g peptone, 20 g dextrose, 20 g agar per liter). As shown in [Additional file 3], samples of the DBY747 WT strain were grown to mid-log phase and treated with doses of agents determined to produce ~20–25% lethality following a 15 min exposure: calicheamicin (1 nM), neocarzinostatin (4 nM), esperamicin A1 (3 nM), and γ-radiation (210 Gy delivered at 105 Gy/min in a 60Co source with 13 min post-irradiation incubation at ambient temperature). Similar studies were performed with the BY4741 strain, with similar results [see Additional file 4]. All exposures were performed at ambient temperature under identical conditions of light and temperature, and cells were snap frozen in liquid nitrogen immediately after the 15 min exposure period.
RNA preparation/cRNA synthesis
Total RNA was extracted from pelleted cells using a hot phenol protocol  that has been used successfully in previous expression profiling studies [5, 9, 10], including one with calicheamicin . AE buffered phenol (50 mM sodium acetate pH 5.3, 10 mM EDTA, pH 8.0) was utilized. RNA was precipitated from the aqueous phase of the phenol extraction. Labeled cRNA was generated as follows. Total RNA was converted into single-stranded cDNA using a modified oligo(dT) primer with a 5' T7 RNA polymerase promoter sequence and reverse transcriptase (SuperScript II RT, Gibco). Double-stranded cDNA was generated using DNA polymerase and DNA ligase (Invitrogen Life Technologies) and purified. Biotin-labeled cRNA was generated using in vitro transcription with T7 RNA polymerase (ENZO BioArray HighYield RNA transcript Labeling Kit, Affymetrix, CA). Reactions were carried out for 5 h at 37°C and cRNA was purified using RNeasy spin columns (Qiagen, CA). cRNA was quantified at UV260 and 15 μg of RNA was fragmented randomly using (200 mM Tris-Acetate, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for 35 min.
GeneChip® hybridizations and image analysis
Hybridizations were performed as follows. Fragmented cRNA was hybridized to GeneChip® (YG-S98, Affymetrix, CA) at a concentration of 0.05 μg/μl in 200 μl of Affymetrix buffer (100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20) with GeneChip® eukaryotic hybridization controls (GeneChip® Eukaryotic Hybridization Controls Kit, Affymetrix, CA) in the presence of 0.1 mg/ml herring sperm DNA and 0.5 mg/ml acetylated BSA at 40°C for 16 h with constant rotation. Arrays were rinsed after hybridization with 200 μl of stringent wash buffer (100 mM MES, 0.1 M NaCl, 0.01% Tween 20) followed by a non-stringent wash (6XSSPE, 0.01% Tween 20). 20XSSPE had the following composition (3 M NaCl, 0.2 M NaH2PO4, 0.02 M EDTA). Staining was done with 2 μg/ml streptavidin-phycoerytherin and 1 mg/ml acetylated BSA in 6xSSPE-T. Arrays were scanned using a HP G2500A GeneArray scanner.
Data pre-processing and differential gene testing
Untreated and treated samples were analyzed in biological triplicate on YG-S98 arrays. Quantile normalization was carried out using the Robust Multichip Average (RMA) algorithm . Transcripts that were absent across all experiments were identified using Absent/Present calls (Microarray Suite 5.0) and filtered for transcripts that were not expressed in any experiment. Differential gene expression was calculated using the dual filtering criteria of ≥1.5 fold change and statistical significance determined through the Local Pooled Error test (LPE)  with an adjustment for false discovery rate calculation of p value of ≤0.10 . All microarray data have been submitted to the Gene Expression Omnibus Database (Series, GSE5301).
Gene Ontology Enrichment Analysis
Statistical evaluation of co-regulated groups of genes was carried out through Gene Ontology Enrichment Analysis within the Functional Specification Database (funspec.med.utoronto.ca) . Co-regulated yeast ORFs were classified according to the Gene Ontology Biological Process and the hypergeometric distribution (p < 0.01) used to assess enrichment of a particular gene category. The hypergeometric distribution asses for each gene ontology category, the probability (p-value) of observing such an overlap by chance is calculated as:
where G is the size of the genome, C is the number of genes in the genome having that attribute, n is the size of the query cluster, of which k are known to possess the attribute.
To identify genes with expression associated with double- or single-strand damage, all modulated ORFs across experimental conditions (225 total) were analyzed for association with induced expression ratio relative to percent single or double-strand lesion formation. Linear regression was performed on expression ratios relative to percent of direct single-strand or double-strand breaks with R2>0.6 and percent of total deoxyribose oxidation events with R2>0.8 selected for associated gene sets (Figure 4).
The authors wish to thank Ms Mary Montesalzo and Dr. Izle Skare at the University of Massachusetts at Lowell for expert assistance with the γ-irradiations. This work was supported by NIH grant CA103146 (PCD). Transcriptional profiling studies were performed in the Genomics and Bioinformatics Core of the MIT Center for Environmental Health Sciences supported by NIEHS grant ES002109. Support for RF and TB was provided by U19-ES11399 and CA55042.
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