A "Hole Punched Plate" method for easy generation and harvesting of microconidia in the dermatophyte Trichophyton rubrum.

Handling of the medically important dermatophyte Trichophyton rubrum in the laboratory typically requires the generation of spores - for storage, treatment and plating when needed. The described method allows technically simple but efficient generation and harvesting of microconidia by cutting holes in Sabouraud dextrose agar medium that is covered by a mature T. rubrum mycelium.

Replicating damaged DNA in eukaryotes.

DNA damage is one of many possible perturbations that challenge the mechanisms that preserve genetic stability during the copying of the eukaryotic genome in S phase. This short review provides, in the first part, a general introduction to the topic and an overview of checkpoint responses. In the second part, the mechanisms of error-free tolerance in response to fork-arresting DNA damage will be discussed in some detail.

Rad5 template switch pathway of DNA damage tolerance determines synergism between cisplatin and NSC109268 in Saccharomyces cerevisiae.

The success of cisplatin (CP) based therapy is often hindered by acquisition of CP resistance. We isolated NSC109268 as a compound altering cellular sensitivity to DNA damaging agents. Previous investigation revealed an enhancement of CP sensitivity by NSC109268 in wild-type Saccharomyces cerevisiae and CP-sensitive and -resistant cancer cell lines that correlated with a slower S phase traversal. Here, we extended these studies to determine the target pathway(s) of NSC109268 in mediating CP sensitization, using yeast as a model. We reasoned that mutants defective in the relevant target of NSC109268 should be hypersensitive to CP and the sensitization effect by NSC109268 should be absent or strongly reduced. A survey of various yeast deletion mutants converged on the Rad5 pathway of DNA damage tolerance by template switching as the likely target pathway of NSC109268 in mediating cellular sensitization to CP. Additionally, cell cycle delays following CP treatment were not synergistically influenced by NSC109268 in the CP hypersensitive rad5Δ mutant. The involvement of the known inhibitory activities of NSC109268 on 20S proteasome and phosphatases 2Cα and 2A was tested. In the CP hypersensitive ptc2Δptc3Δpph3Δ yeast strain, deficient for 2C and 2A-type phosphatases, cellular sensitization to CP by NSC109268 was greatly reduced. It is therefore suggested that NSC109268 affects CP sensitivity by inhibiting the activity of unknown protein(s) whose dephosphorylation is required for the template switch pathway.

Role of polymerase η in mitochondrial mutagenesis of Saccharomyces cerevisiae.

DNA polymerase η mostly catalyzes an error-free bypass of the most frequent UV lesions, pyrimidine dimers of the cyclobutane-type. In addition to its nuclear localization, we show here for the first time its mitochondrial localization in budding yeast. In mitochondria, this polymerase improves bypass replication fidelity opposite UV damage as shown in base pair substitution and frameshift assays. For base pair substitutions, polymerase η appears to be related in function and epistatic to DNA polymerase ζ which, however, plays the opposite role in the nucleus.

Saccharomyces cerevisiae Tel2 plays roles in TORC signaling and telomere maintenance that can be mutationally separated.

The evolutionary conserved Tel2 protein appears to function as a co-chaperone required for the activity of phosphatidylinositol 3-kinase-like protein kinases (PIKKs). Since Saccharomyces cerevisiae Tel2 is essential for viability and only a single point mutant (Tel2-1) had been characterized so far, the possible range of phenotypes associated with Tel2 mutations was unknown. We used random in vitro mutagenesis and plasmid shuffling to create additional point mutants. No significant sensitivity towards DNA damaging agents or hydroxyurea was evident, indicating that Tel2 is not required for Mec1 function. However, as frequent novel phenotypes, we detected slow growth or enhanced lethality in response to rapamycin that could be correlated with lower level and activity of Tor1 or of both Tor1 and Tor2, respectively. The newly isolated mutant with the most severe phenotype, Tel2-13, is comprised of 8 amino acid changes. Two mutated residues of Tel2-13 near the N-terminus and close to Tel2-1 are sufficient for shortened telomeres whereas multiple mutations within the C-terminal two thirds of the protein are required for enhanced rapamycin lethality. Our findings demonstrate separation of function explainable by differential binding of Tel2 to its PIKK substrates Tel1 or Tor1/Tor2 and thus a critical contribution of Tel2 to the interface that links various PIKKs to this chaperone complex.

Phenotypes associated with Saccharomyces cerevisiae Hug1 protein, a putative negative regulator of dNTP Levels, reveal similarities and differences with sequence-related Dif1.

Saccharomyces cerevisiae Hugl is a small protein of unknown function that is highly inducible following replication stress and DNA damage. Its deletion suppresses the lethality of deletion of checkpoint kinase Mecl. Although DNA damage responses were largely normal in the HUG1 deletion mutant, we found enhanced resistance towards heat in logarithmic phase. In response to simultaneous carbon and replication stress, overall growth delay and less pseudohyphal filament formation were evident. These novel phenotypes are shared with deletion mutants of the negative regulators of ribonucleotide reductase, Difl and Smll. Microarray analysis showed the influence of Hugl on the expression of a large number of transcripts, including stress-related transcripts. Elevated dNTP levels in hugl Δ cells may result in a stress response reflected by the observed phenotypes and transcript profiles. However, in contrast to a deletion of structurally related Difl, Rnr2-Rnr4 subcellular localization is not grossly altered in a Hugl deletion mutant. Thus, although Hugl appears to be derived from the Rnr2-Rnr4 binding region of Difl, its mechanism of action must be independent of determining the localization of Rnr2-Rnr4.

NSC109268 potentiates cisplatin-induced cell death in a p53-independent manner.

BACKGROUND: Ovarian cancer is the leading cause of death among gynecological cancers. Cisplatin is one of the most effective anticancer drugs used in the treatment of ovarian cancer. Development of resistance to cisplatin limits its therapeutic use. Most of the anticancer drugs, including cisplatin, are believed to kill cancer cells by inducing apoptosis and a defect in apoptotic signaling can contribute to drug resistance. The tumor suppressor protein p53 plays a critical role in DNA damage-induced apoptosis. During a yeast-based drug screening, NSC109268 was identified to enhance cellular sensitivity to cisplatin. The objective of the present study is to determine if p53 is responsible for cisplatin sensitization by NSC109268. RESULTS: NSC109268 enhanced sensitivity of ovarian cancer 2008 cells and its cisplatin resistant counterpart 2008/C13* cells which express wild-type p53. The potentiation of cisplatin sensitivity by NSC109268 was greater in 2008/C13* cells compared to 2008 cells. Cisplatin caused a concentration-dependent increase in p53 in 2008 and 2008/C13* cells, and the induction of p53 correlated with cisplatin-induced apoptosis as determined by the cleavage of PARP. NSC109268 alone had no effect on p53 but it enhanced p53 level in response to cisplatin. Knockdown of p53 by siRNA, however, did not attenuate cell death in response to cisplatin or combination of NSC109268 and cisplatin. CONCLUSIONS: These results demonstrate that NSC109268 enhances sensitivity of ovarian cancer 2008 cells to cisplatin independent of p53.

Enhancement of cisplatin sensitivity by NSC109268 in budding yeast and human cancer cells is associated with inhibition of S-phase progression.

PURPOSE: NSC109268 has been described previously as inhibitor of proteasomal degradation and of phosphatase 2Calpha. In a yeast screen, we isolated NSC109268 as an agent altering sensitivity to DNA-damaging agents. We found that NSC109268 and the related compound NSC109272 enhance cellular sensitivity to cis- and transplatin but reduce sensitivity to nitrogen mustard. We explored if similar effects could be found in human cancer cells and if cell cycle analysis could hint at the underlying molecular mechanism. METHODS: Haploid yeast cells were treated in suspension with platinum agents and nitrogen mustard alone or in combination with NSC compounds, and survival was measured by colony-formation assays. Sensitivity of ovarian and prostate cancer cells toward these treatments was evaluated using the MTS assay. Cell cycle progression was determined by flow cytometry. RESULTS: The enhancement of cisplatin sensitivity by NSC109268 found in yeast was confirmed in cisplatin-sensitive and cisplatin-resistant human ovarian cancer lines and in prostate cancer cells. In yeast and in human carcinoma cells, a correlation of enhanced sensitivity with delaying S-phase progression was revealed. CONCLUSION: The known activities of NSC109268 as proteasome or phosphatase inhibitor could explain the phenotype of S-phase delay by assuming a higher initial DNA damage load, inhibition of DNA translesion synthesis or extended checkpoint arrest.

Checkpoint kinase phosphorylation in response to endogenous oxidative DNA damage in repair-deficient stationary-phase Saccharomyces cerevisiae.

Stationary-phase Saccharomyces cerevisiae can serve as a model for post-mitotic cells of higher eukaryotes. Phosphorylation and activation of the checkpoint kinase Rad53 was observed after more than 2 days of culture if two major pathways of oxidative DNA damage repair, base excision repair (BER) and nucleotide excision repair (NER), are inactive. The wild type showed a low degree of Rad53 phosphorylation when the incubation period was drastically increased. In the ber ner strain, Rad53 phosphorylation can be abolished by inclusion of antioxidants or exclusion of oxygen. Furthermore, this modification and enhanced mutagenesis in extended stationary phase were absent in rho degrees strains, lacking detectable mitochondrial DNA. This checkpoint response is therefore thought to be dependent on reactive oxygen species originating from mitochondrial respiration. There was no evidence for progressive overall telomere shortening during stationary-phase incubation. Since Rad50 (of the MRN complex) and Mec1 (the homolog of ATR) were absolutely required for the observed checkpoint response, we assume that resected random double-strand breaks are the critical lesion. Single-strand resection may be accelerated by unrepaired oxidative base damage in the vicinity of a double-strand break.

SBF transcription factor complex positively regulates UV mutagenesis in Saccharomyces cerevisiae.

The collection of gene deletion mutants of Saccharomyces cerevisiae was used to screen for novel genes required for UV-induced mutagenesis. We found the SBF transcription factor (Swi4/Swi6 protein complex) to be required for wild-type levels of UV mutability in forward and reverse mutation assays. Expression of translesion polymerase zeta component Rev7 was identified as a target of SBF-dependent regulation.

The available SRL3 deletion strain of Saccharomyces cerevisiae contains a truncation of DNA damage tolerance protein Mms2: Implications for Srl3 and Mms2 functions.

A screen of the commercially available collection of haploid deletion mutants of Saccharomyces cerevisiae for spontaneous mutator mutants newly identified a deletion of SRL3. This gene had been previously isolated as a suppressor of lethality of checkpoint kinase deletions if overexpressed. We found DNA damage sensitivity and extended checkpoint arrests to be associated with this strain. However, when crossed to wild-type, a mutant gene conferring these phenotypes was found to segregate from the SRL3 deletion. The mutation was identified as a C-terminal truncation of Mms2, an E2 ubiquitin conjugating enzyme involved in error-free replicative bypass of lesions. This confirmed an earlier report that Mms2 may be required to restrain error-prone polymerase ζ activity and underscored that residues of the C-terminus are necessary for Mms2 function. Srl3, on the other hand, does not appear to influence DNA damage sensitivity or spontaneous mutability if deleted. However, the absence of these phenotypes does not contradict its likely role as a positive regulator of dNTP levels.

Regulation of Saccharomyces cerevisiae DNA polymerase eta transcript and protein.

RAD30-encoded DNA polymerase eta functions as a translesion polymerase that can bypass the most frequent types of UV-induced pyrimidine photoproducts in an error-free manner. Although its transcript is UV-inducible in Saccharomyces cerevisiae, Rad30 (studied as a Rad30-Myc fusion) is a stable protein whose levels do not fluctuate following UV treatment or during cell cycle progression. Rad30 protein is subject to monoubiquitination whose level is upregulated in G1 and downregulated during S-phase reentry. This downregulation is accelerated in UV-treated cells. A missense mutation (L577Q) of the ubiquitin binding domain (UBZ) confers a reduced degree of ubiquitination outside of G1 and a complete failure to stably interact with ubiquitinated substrates. This mutation confers a phenotype resembling a complete RAD30 deletion, thus attesting to the significance of the UBZ motif for polymerase eta function in vivo.

Roles of Saccharomyces cerevisiae RAD17 and CHK1 checkpoint genes in the repair of double-strand breaks in cycling cells.

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.

Characterization of checkpoint responses to DNA damage in Saccharomyces cerevisiae: basic protocols.

In spite of certain special features of its cell cycle, the yeast Saccharomyces cerevisiae has proved to be an excellent and widely used model to study eukaryotic checkpoint responses to DNA damage. This chapter primarily summarizes selected cytological methods that are useful for initial characterization of cell cycle responses. These can be useful in order to study mutants, conditions, or selected DNA damaging agents and experimental examples are given. We have also included protocols for flow-cytometric cell cycle analysis and for determination of Rad53 phosphorylation, a commonly used indicator of checkpoint activation.

Mutagenic effects of abasic and oxidized abasic lesions in Saccharomyces cerevisiae.

2-deoxyribonolactone (L) and 2-deoxyribose (AP) are abasic sites that are produced by ionizing radiation, reactive oxygen species and a variety of DNA damaging agents. The biological processing of the AP site has been examined in the yeast Saccharomyces cerevisiae. However, nothing is known about how L is processed in this organism. We determined the bypass and mutagenic specificity of DNA containing an abasic site (AP and L) or the AP analog tetrahydrofuran (F) using an oligonucleotide transformation assay. The tetrahydrofuran analog and L were bypassed at 10-fold higher frequencies than the AP lesions. Bypass frequencies of lesions were greatly reduced in the absence of Rev1 or Polzeta (rev3 mutant), but were only marginally reduced in the absence of Poleta (rad30 mutant). Deoxycytidine was the preferred nucleotide inserted opposite an AP site whereas dA and dC were inserted at equal frequencies opposite F and L sites. In the rev1 and rev3 strains, dA was the predominant nucleotide inserted opposite these lesions. Overall, we conclude that both Rev1 and Polzeta are required for the efficient bypass of abasic sites in yeast.

The conserved Mec1/Rad53 nuclear checkpoint pathway regulates mitochondrial DNA copy number in Saccharomyces cerevisiae.

How mitochondrial DNA (mtDNA) copy number is determined and modulated according to cellular demands is largely unknown. Our previous investigations of the related DNA helicases Pif1p and Rrm3p uncovered a role for these factors and the conserved Mec1/Rad53 nuclear checkpoint pathway in mtDNA mutagenesis and stability in Saccharomyces cerevisiae. Here, we demonstrate another novel function of this pathway in the regulation of mtDNA copy number. Deletion of RRM3 or SML1, or overexpression of RNR1, which recapitulates Mec1/Rad53 pathway activation, resulted in an approximately twofold increase in mtDNA content relative to the corresponding wild-type yeast strains. In addition, deletion of RRM3 or SML1 fully rescued the approximately 50% depletion of mtDNA observed in a pif1 null strain. Furthermore, deletion of SML1 was shown to be epistatic to both a rad53 and an rrm3 null mutation, placing these three genes in the same genetic pathway of mtDNA copy number regulation. Finally, increased mtDNA copy number via the Mec1/Rad53 pathway could occur independently of Abf2p, an mtDNA-binding protein that, like its metazoan homologues, is implicated in mtDNA copy number control. Together, these results indicate that signaling through the Mec1/Rad53 pathway increases mtDNA copy number by altering deoxyribonucleoside triphosphate pools through the activity of ribonucleotide reductase. This comprises the first linkage of a conserved signaling pathway to the regulation of mitochondrial genome copy number and suggests that homologous pathways in humans may likewise regulate mtDNA content under physiological conditions.

DNA decay and limited Rad53 activation after liquid holding of UV-treated nucleotide excision repair deficient S. cerevisiae cells.

The DNA damage checkpoint is a surveillance mechanism activated by DNA lesions and devoted to the maintenance of genome stability. It is considered as a signal transduction cascade, involving a sensing step, the activation of a set of protein kinases and the transmission and amplification of the damage signal through several phosphorylation events. In budding yeast many players of this pathway have been identified. Recent work showed that G1 and G2 checkpoint activation in response to UV irradiation requires prior recognition and processing of UV lesions by nucleotide excision repair (NER) factors that likely recruit checkpoint proteins near the damage. However, another report suggested that NER was not required for checkpoint function. Since the functional relationship between repair mechanisms and checkpoint activation is a very important issue in the field, we analyzed, under different experimental conditions, whether lesion processing by NER is required for checkpoint activation. We found that DNA damage checkpoint can be triggered in an NER-independent manner only if cells are subjected to liquid holding after UV treatment. This incubation causes a time-dependent breakage of DNA strands in NER-deficient cells and leads to partial activation of the checkpoint kinase. The analysis of the genetic requirements for this alternative activation pathway suggest that it requires Mec1 and the Rad17 complex and that the observed DNA breaks are likely to be due to spontaneous decay of damaged DNA.

Spontaneous DNA damage in Saccharomyces cerevisiae elicits phenotypic properties similar to cancer cells.

To determine the spectrum of effects elicited by specific levels of spontaneous DNA damage, a series of isogenic Saccharomyces cerevisiae strains defective in base excision repair (BER) and nucleotide excision repair (NER) were analyzed. In log phase of growth, when compared with wild type (WT) or NER-defective cells, BER-defective cells and BER/NER-defective cells possess elevated levels of unrepaired, spontaneous oxidative DNA damage. This system allowed establishment of a range of approximately 400 to 1400 Ntg1p-recognized DNA lesions per genome necessary to provoke profound biological changes similar in many respects to the phenotypic properties of cancer cells. The BER/NER-defective cells are genetically unstable, exhibiting mutator and hyper-recombinogenic phenotypes. They also exhibit aberrations in morphology, DNA content, and growth characteristics compared with WT, BER-defective, and NER-defective cells. The BER/NER-defective cells also possess increased levels of intracellular reactive oxygen species, activate the yeast checkpoint response pathway via Rad53p phosphorylation in stationary phase, and show profound changes in transcription patterns, a subset of which can be ascribed to responses resulting from unrepaired DNA damage. By establishing a relationship between specific levels of spontaneous DNA damage and the ensuing deleterious biological consequences, these yeast DNA excision repair-defective strains are an informative model for gauging the progressive biological consequences of spontaneous DNA damage accumulation and may have relevancy for delineating underlying mechanisms in tumorigenesis.