The Saccharomyces cerevisiae PDS1 and RAD9 checkpoint genes control different DNA double-strand break repair pathways

In response to DNA damage, the Saccharomyces cerevisiae securin Pds1 blocks anaphase promotion by inhibiting ESP1-dependent degradation of cohesins. PDS1 is positioned downstream of the MEC1- and RAD9-mediated DNA damage-induced signal transduction pathways. Because cohesins participate in postreplicative repair and the pds1 mutant is radiation sensitive, we identified DNA repair pathways that are PDS1-dependent. We compared the radiation sensitivities and recombination phenotypes of pds1, rad9, rad51 single and double mutants, and found that whereas pds1 rad9 double mutants were synergistically more radiation sensitive than single mutants, pds1 rad51 mutants were not. To determine the role of PDS1 in recombinational repair pathways, we measured spontaneous and DNA damage-associated sister chromatid exchanges (SCEs) after exposure to X rays, UV and methyl methanesulfonate (MMS) and after the initiation of an HO endonuclease-generated double-strand break (DSB). The rates of spontaneous SCE and frequencies of DNA damage-associated SCE were similar in wild type and pds1 strains, but the latter exhibited reduced viability after exposure to DNA damaging agents. To determine whether pds1 mutants were defective in other pathways for DSB repair, we measured both single-strand annealing (SSA) and non-homologous end joining (NHEJ) in pds1 mutants. We found that the pds1 mutant was defective in SSA but efficient at ligating cohesive ends present on a linear plasmid. We therefore suggest that checkpoint genes control different pathways for DSB repair, and PDS1 and RAD9 have different roles in recombinational repair.     

Histone H3 lysine 56 acetylation and the response to DNA replication fork damage

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.     

Genome-wide analysis of barcoded Saccharomyces cerevisiae gene-deletion mutants in pooled cultures

The availability of a near-complete (96%) collection of gene-deletion mutants in Saccharomyces cerevisiae greatly facilitates the systematic analyses of gene function in yeast. The unique 20 bp DNA 'barcodes' or 'tags' in each deletion strain enable the individual fitness of thousands of deletion mutants to be resolved from a single pooled culture. Here, we present protocols for the study of pooled cultures of tagged yeast deletion mutants with a tag microarray. This process involves five main steps: pooled growth, isolation of genomic DNA, PCR amplification of the barcodes, array hybridization and data analysis. Pooled deletion screening can be used to study gene function, uncover a compound's mode of action and identify drug targets. In addition to these applications, the general method of studying pooled samples with barcode arrays can also be adapted for use with other types of samples, such as mutant collections in other organisms, short interfering RNA vectors and molecular inversion probes.     

Chl1 and Ctf4 are required for damage-induced recombinations

Deletion mutants of CHL1 or CTF4, which are required for sister chromatid cohesion, showed higher sensitivity to the DNA damaging agents methyl methanesulfonate (MMS), hydroxyurea (HU), phleomycin, and camptothecin, similar to the phenotype of mutants of RAD52, which is essential for recombination repair. The levels of Chl1 and Ctf4 associated with chromatin increased considerably after exposure of the cells to MMS and phleomycin. Although the activation of DNA damage checkpoint did not affected in chl1 and ctf4 mutants, the repair of damaged chromosome was inefficient, suggesting that Chl1 and Ctf4 act in DNA repair. In addition, MMS-induced sister chromatid recombination in haploid cells, and, more importantly, MMS-induced recombination between homologous chromosomes in diploid cells were impaired in these mutants. Our results suggest that Chl1 and Ctf4 are directly involved in homologous recombination repair rather than acting indirectly via the establishment of sister chromatid cohesion.     

Histone H1 variant, H1R is involved in DNA damage response

In Saccharomyces cerevisiae, the linker histone HHO1 is involved in DNA repair. In higher eukaryotes, multiple variants of linker histone H1 exist but their involvement in the DNA damage response is unknown. To address this issue, we examined sensitivity to genotoxic agents in chicken DT40 cells lacking specific H1 variants. Among the six H1 variant mutants, only H1R(-/-) DT40 cells exhibited increased sensitivity to the alkylating agent methyl-methanesulfonate (MMS). The MMS sensitivity of H1R(-/-) cells was not enhanced by inactivation of Rad54. H1R(-/-) DT40 cells also exhibited: (i) a reduction in gene targeting efficiencies, (ii) impaired sister chromatid exchange, and (iii) an accumulation of IR-induced chromosomal aberrations at the G2 phase, all of which indicate the involvement of H1R in the Rad54-mediated homologous recombination (HR) pathway. The mobility of H1R but not H1L in the nucleus decreased after MMS treatment and the repair of double-stranded breaks generated by I-SceI was unaffected in H1R(-/-) cells, suggesting that H1R integrates into HR-mediated repair pathways at the chromosome structure level. Together, these findings provide the first genetic evidence that a specific H1 variant plays a unique and important role in the DNA damage response in vertebrates.     

Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p

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.     

Yeast Barcoders: a chemogenomic application of a universal donor-strain collection carrying bar-code identifiers

The ability to perform complex bioassays in parallel enables experiments that are otherwise impossible because of throughput and cost constraints. For example, highly parallel chemical-genetic screens using pooled collections of thousands of defined Saccharomyces cerevisiae gene deletion strains are feasible because each strain is bar-coded with unique DNA sequences. It is, however, time-consuming and expensive to individually bar-code individual strains. To provide a simple and general method of barcoding yeast collections, we built a set of donor strains, called Barcoders, with unique bar codes that can be systematically transferred to any S. cerevisiae collection. We applied this technology by generating a collection of bar-coded 'decreased abundance by mRNA perturbation' (DAmP) loss-of-function strains comprising 87.1% of all essential yeast genes. These experiments validate both the Barcoders and the DAmP strain collection as useful tools for genome-wide chemical-genetic assays.     

The ability of Sgs1 to interact with DNA topoisomerase III is essential for damage-induced recombination

SGS1 encodes a protein having DNA helicase activity, and a mutant allele of SGS1 was identified as a suppressor of the slow growth phenotype of top3 mutants. In this study, we examined whether Sgs1 prevents formation of DNA double strand breaks (DSBs) or is involved in DSB repair following exposure to methyl methanesulfonate (MMS). An analysis by pulsed-field gel electrophoresis and epistasis analyses indicated that Sgs1 is required for DSB repair that involves Rad52. In addition, analyses on the relationship between Sgs1 and proteins involved in DSB repair suggested that Sgs1 and Mre11 function via independent pathways both of which require Rad52. In sgs1 mutants, interchromosomal heteroallelic recombination and sister chromatid recombination (SCR) were not induced upon exposure to MMS, though both were induced in wild type cells, indicating the involvement of Sgs1 in heteroallelic recombination and SCR. Surprisingly, the ability of Sgs1 to bind to DNA topoisomerase III (Top3) was absolutely required for the induction of heteroallelic recombination and SCR and suppression of MMS sensitivity but its helicase activity was not, suggesting that Top3 plays a more important role in both recombinations than the DNA helicase activity of Sgs1.     

Systems based mapping demonstrates that recovery from alkylation damage requires DNA repair, RNA processing, and translation associated networks

The identification of cellular responses to damage can promote mechanistic insight into stress signalling. We have screened a library of 3968 Escherichia coli gene-deletion mutants to identify 99 gene products that modulate the toxicity of the alkylating agent methyl methanesulfonate (MMS). We have developed an ontology mapping approach to identify functional categories over-represented with MMS-toxicity modulating proteins and demonstrate that, in addition to DNA re-synthesis (replication, recombination, and repair), proteins involved in mRNA processing and translation influence viability after MMS damage. We have also mapped our MMS-toxicity modulating proteins onto an E. coli protein interactome and identified a sub-network consisting of 32 proteins functioning in DNA repair, mRNA processing, and translation. Clustering coefficient analysis identified seven highly connected MMS-toxicity modulating proteins associated with translation and mRNA processing, with the high connectivity suggestive of a coordinated response. Corresponding results from reporter assays support the idea that the SOS response is influenced by activities associated with the mRNA-translation interface.     

甲磺酸甲酯对酵母DNA损伤及端粒酶活性的影响

观察甲磺酸甲酯 (MMS)对酿酒酵母S2 88C细胞染色体DNA的损伤及端粒酶活性的调节。结果表明 ,甲磺酸甲酯引起酵母细胞DNA损伤 ,随着MMS浓度的增加及作用时间的延长 ,DNA损伤程度加重 ,同时明显提高酵母细胞端粒酶活性。当用 0 .4mmol/LMMS作用 72h后 ,端粒酶活性最高 (是对照组的1.4 7倍 ) ,在作用 96h及 12 0h后端粒酶活性逐渐下降 ,但均高于对照组。甲磺酸甲酯对酿酒酵母S2 88C细胞端粒酶活性的上调作用可能与其DNA损伤有关 ,断裂DNA的损伤后修复可能是端粒酶介导的。     

A new Schizosaccharomyces pombe base excision repair mutant,nth1, reveals overlapping pathways for repair of DNA base damage

Endonuclease III (Nth) enzyme from Escherichia coli is involved in base excision repair of oxidised pyrimidine residues in DNA. The Schizosaccharomyces pombe Nth1 protein is a sequence and functional homologue of E. coli Nth, possessing both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activity. Here, we report the construction and characterization of the S. pombe nth1 mutant. The nth1 mutant exhibited no enhanced sensitivity to oxidising agents, UV or gamma-irradiation, but was hypersensitive to the alkylating agent methyl methanesulphonate (MMS). Analysis of base excision from DNA exposed to [3H]methyl-N-nitrosourea showed that the purified Nth1 enzyme did not remove alkylated bases such as 3-methyladenine and 7-methylguanine whereas methyl-formamidopyrimidine was excised efficiently. The repair of AP sites in S. pombe has previously been shown to be independent of Apn1-like AP endonuclease activity, and the main reason for the MMS sensitivity of nth1 cells appears to be their lack of AP lyase activity. The nth1 mutant also exhibited elevated frequencies of spontaneous mitotic intrachromosomal recombination, which is a phenotype shared by the MMS-hypersensitive DNA repair mutants rad2, rhp55 and NER repair mutants rad16, rhp14, rad13 and swi10. Epistasis analyses of nth1 and these DNA repair mutants suggest that several DNA damage repair/tolerance pathways participate in the processing of alkylation and spontaneous DNA damage in S. pombe.     

Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.     

Induction of genome instability by DNA damage in Saccharomyces cerevisiae

The accumulation of gross chromosomal rearrangements (GCRs) is a characteristic of many types of cancer cells, although it is unclear what defects cause these rearrangements and how the different types of GCRs observed are formed. In the present study, we have used a Saccharomyces cerevisiae system for measuring GCRs to analyze the ability of a variety of DNA damaging agents to induce GCRs. The two most potent inducers of GCRs observed were methyl methane sulfonate (MMS) and HO-endonuclease-induced double strand breaks (DSBs). Bleomycin, camptothecan and gamma-irradiation induced intermediate levels of GCRs and cisplatin induced very low levels of GCRs whereas N-methyl-NPRIME;-nitro-N-nitrosoguanidine (MNNG) and ethyl methane sulfonate (EMS) primarily induced base substitution mutations. MMS treatment primarily induced rearrangements in which the end of a chromosome was deleted and a new telomere was added (telomere additions) and also induced translocations. Consistent with this GCR spectrum, the formation of MMS-induced GCRs was primarily dependent on telomere maintenance functions and were completely eliminated in mutants that were defective for both telomere maintenance functions and non-homologous end joining (NHEJ). In contrast, HO-endonuclease DSBs induced mostly translocations and interstitial deletions whereas few telomere additions were observed. Genetic analysis indicated that HO DSB-induced GCRs were suppressed by a number of pathways including the DNA damage checkpoints, DSB repair pathways and NHEJ.     

Glucose-induced gradual phenotypic modulation of cultured human glomerular epithelial cells may be independent of Wilms’ tumor 1 (WT1)

Background

Melatonin, a hormone-like substance involved in the regulation of the circadian rhythm, has been demonstrated to protect cells against oxidative DNA damage and to inhibit tumorigenesis.

Results

In the current study, we investigated the effect of melatonin on DNA strand breaks using the alkaline DNA comet assay in breast cancer (MCF-7) and colon cancer (HCT-15) cell lines. Our results demonstrated that cells pretreated with melatonin had significantly shorter Olive tail moments compared to non-melatonin treated cells upon mutagen (methyl methanesulfonate, MMS) exposure, indicating an increased DNA repair capacity after melatonin treatment. We further examined the genome-wide gene expression in melatonin pretreated MCF-7 cells upon carcinogen exposure and detected altered expression of many genes involved in multiple DNA damage responsive pathways. Genes exhibiting altered expression were further analyzed for functional interrelatedness using network- and pathway-based bioinformatics analysis. The top functional network was defined as having relevance for “DNA Replication, Recombination, and Repair, Gene Expression, [and] Cancer”.

Conclusions

These findings suggest that melatonin may enhance DNA repair capacity by affecting several key genes involved in DNA damage responsive pathways.     

The 70 kDa subunit of replication protein A is required for the G1/S and intra-S DNA damage checkpoints in budding yeast.

The rfa1-M2 and rfa1-M4 Saccharomyces cerevisiae mutants, which are altered in the 70 kDa subunit of replication protein A (RPA) and sensitive to UV and methyl methane sulfonate (MMS), have been analyzed for possible checkpoint defects. The G1/S and intra-S DNA damage checkpoints are defective in the rfa1-M2 mutant, since rfa1-M2 cells fail to properly delay cell cycle progression in response to UV irradiation in G1 and MMS treatment during S phase. Conversely, the G2/M DNA damage checkpoint and the S/M checkpoint are proficient in rfa1-M2 cells and all the checkpoints tested are functional in the rfa1-M4 mutant. Preventing S phase entry by alpha-factor treatment after UV irradiation in G1 does not change rfa1-M4 cell lethality, while it allows partial recovery of rfa1-M2 cell viability. Therefore, the hypersensitivity to UV and MMS treatments observed in the rfa1-M4 mutant might only be due to impairment of RPA function in DNA repair, while the rfa1-M2 mutation seems to affect both the DNA repair and checkpoint functions of Rpa70.     

XRCC1 is required for DNA single-strand break repair in human cells

The X-ray repair cross complementing 1 (XRCC1) protein is required for viability and efficient repair of DNA single-strand breaks (SSBs) in rodents. XRCC1-deficient mouse or hamster cells are hypersensitive to DNA damaging agents generating SSBs and display genetic instability after such DNA damage. The presence of certain polymorphisms in the human XRCC1 gene has been associated with altered cancer risk, but the role of XRCC1 in SSB repair (SSBR) in human cells is poorly defined. To elucidate this role, we used RNA interference to modulate XRCC1 protein levels in human cell lines. A reduction in XRCC1 protein levels resulted in decreased SSBR capacity as measured by the comet assay and intracellular NAD(P)H levels, hypersensitivity to the cell killing effects of the DNA damaging agents methyl methanesulfonate (MMS), hydrogen peroxide and ionizing radiation and enhanced formation of micronuclei following exposure to MMS. Lowered XRCC1 protein levels were also associated with a significant delay in S-phase progression after exposure to MMS. These data clearly demonstrate that XRCC1 is required for efficient SSBR and genomic stability in human cells.