Roland Chanet

Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins.

Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses.

Regulation of the Saccharomyces cerevisiae Srs2 helicase during the mitotic cell cycle, meiosis and after irradiation.

The expression of theSRS2 gene, which encodes a DNA helicase involved in DNA repair inSaccharomyces cerevisiae, was studied using anSRS2-lacZ fusion integrated at the chromosomalSRS2 locus. It is shown here that this gene is expressed at a low level and is tightly regulated. It is cell-cycle regulated, with induction probably being coordinated with that of the DNA-synthesis genes, which are transcribed at the G1-S boundary. It is also induced by DNA-damaging agents, but only during the G2 phase of the cell cycle; this distinguishes it from a number of other repair genes, which are inducible throughout the cycle. During meiosis, the expression ofSRS2 rises at a time nearly coincident with commitment to recombination. Sincesrs2 null mutants are radiation sensitive essentially when treated in G1, the mitotic regulation pattern described here leads us to postulate that either secondary regulatory events limit Srs2 activity to G1 cells or Srs2 functions in a repair mechanism associated with replication.

Characterization of mutations that are synthetic lethal with pol3-13, a mutated allele of DNA polymerase delta in Saccharomyces cerevisiae

Abstract The pol3-13 mutation is located in the C-terminal end of POL3, the gene encoding the catalytic subunit of polymerase δ, and confers thermosensitivity onto the Saccharomyces cerevisiae mutant strain. To get insight about DNA replication control, we performed a genetic screen to identify genes that are synthetic lethal with pol3-13. Mutations in genes encoding the two other subunits of DNA polymerase δ (HYS2, POL32) were identified. Mutations in two recombination genes (RAD50, RAD51) were also identified, confirming that homologous recombination is necessary for pol3-13 mutant strain survival. Other mutations were identified in genes involved in repair and genome stability (MET18/MMS19), in the control of origin-firing and/or transcription (ABF1, SRB7), in the S/G2 checkpoint (RAD53), in the Ras-cAMP signal transduction pathway (MKS1), in nuclear pore metabolism (SEH1), in protein degradation (DOC1) and in folding (YDJ1). Finally, mutations in three genes of unknown function were isolated (NBP35, DRE2, TAH18). Synthetic lethality between pol3-13 and each of the three mutants pol32, mms19 and doc1 could be suppressed by a rad18 deletion, suggesting an important role of ubiquitination in DNA replication control. We propose that the pol3-13 mutant generates replicative problems that need both homologous recombination and an intact checkpoint machinery to be overcome.

Redox-sensitive YFP sensors monitor dynamic nuclear and cytosolic glutathione redox changes.

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. To better characterize redox control in the nucleus, we targeted a yellow fluorescent protein-based redox sensor (rxYFP) to the nucleus of the yeast Saccharomyces cerevisiae. Parallel analyses of the redox state of nucleus-rxYFP and cytosol-rxYFP allowed us to monitor distinctively dynamic glutathione (GSH) redox changes within these two compartments under a given condition. We observed that the nuclear GSH redox environment is highly reducing and similar to the cytosol under steady-state conditions. Furthermore, these sensors are able to detect redox variations specific for their respective compartments in glutathione reductase (Glr1) and thioredoxin pathway (Trr1, Trx1, Trx2) mutants that have altered subcellular redox environments. Our mutant redox data provide in vivo evidence that glutathione and the thioredoxin redox systems have distinct but overlapping functions in controlling subcellular redox environments. We also monitored the dynamic response of nucleus-rxYFP and cytosol-rxYFP to GSH depletion and to exogenous low and high doses of H₂O₂ bursts. These observations indicate a rapid and almost simultaneous oxidation of both nucleus-rxYFP and cytosol-rxYFP, highlighting the robustness of the rxYFP sensors in measuring real-time compartmental redox changes. Taken together, our data suggest that the highly reduced yeast nuclear and cytosolic redox states are maintained independently to some extent and under distinct but subtle redox regulation. Nucleus- and cytosol-rxYFP register compartment-specific localized redox fluctuations that may involve exchange of reduced and/or oxidized glutathione between these two compartments. Finally, we confirmed that GSH depletion has profound effects on mitochondrial genome stability but little effect on nuclear genome stability, thereby emphasizing that the critical requirement for GSH during growth is linked to a mitochondria-dependent process.

Potential DNA-binding domains in the RAD18 gene product of Saccharomyces cerevisiae

The RAD18 gene of Saccharomyces cerevisiae is involved in the error-prone DNA repair. Its nucleotide sequence, as reported here, predicts an open reading frame of 1461 nt which corresponds to a protein of 487 amino acids, with an Mr of 55,237. This protein has three putative zinc fingers, two acidic regions and a nucleotide-binding domain, suggesting that it is a nucleic acid-binding protein with a possible regulatory role.

A New Saccharomyces cerevisiae Strain with a Mutant Smt3-Deconjugating Ulp1 Protein Is Affected in DNA Replication and Requires Srs2 and Homologous Recombination for Its Viability

ABSTRACT The Saccharomyces cerevisiae Srs2 protein is involved in DNA repair and recombination. In order to gain better insight into the roles of Srs2, we performed a screen to identify mutations that are synthetically lethal with an srs2 deletion. One of them is a mutated allele of the ULP1 gene that encodes a protease specifically cleaving Smt3-protein conjugates. This allele, ulp1-I615N, is responsible for an accumulation of Smt3-conjugated proteins. The mutant is unable to grow at 37°C. At permissive temperatures, it still shows severe growth defects together with a strong hyperrecombination phenotype and is impaired in meiosis. Genetic interactions between ulp1 and mutations that affect different repair pathways indicated that the RAD51-dependent homologous recombination mechanism, but not excision resynthesis, translesion synthesis, or nonhomologous end-joining processes, is required for the viability of the mutant. Thus, both Srs2, believed to negatively control homologous recombination, and the process of recombination per se are essential for the viability of the ulp1 mutant. Upon replication, mutant cells accumulate single-stranded DNA interruptions. These structures are believed to generate different recombination intermediates. Some of them are fixed by recombination, and others require Srs2 to be reversed and fixed by an alternate pathway.

Redox-sensitive YFP sensors for monitoring dynamic compartment-specific glutathione redox state

Intracellular redox homeostasis is crucial for many cellular functions but accurate measurements of cellular compartment-specific redox states remain technically challenging. Genetically encoded biosensors including the glutathione-specific redox-sensitive yellow fluorescent protein (rxYFP) may provide an alternative way to overcome the limitations of conventional glutathione/glutathione disulfide (GSH/GSSG) redox measurements. This study describes the use of rxYFP sensors for investigating compartment-specific steady redox state and their dynamics in response to stress in human cells. RxYFP expressed in the cytosol, nucleus, or mitochondrial matrix of HeLa cells was responsive to the intracellular redox state changes induced by reducing as well as oxidizing agents. Compartment-targeted rxYFP sensors were able to detect different steady-state redox conditions among the cytosol, nucleus, and mitochondrial matrix. These sensors expressed in human epidermal keratinocytes HEK001 responded to stress induced by ultraviolet A radiation in a dose-dependent manner. Furthermore, rxYFP sensors were able to sense dynamic and compartment-specific redox changes caused by 100 μM hydrogen peroxide (H2O2). Mitochondrial matrix-targeted rxYFP displayed a greater dynamics of oxidation in response to a H2O2 challenge than the cytosol- and nucleus-targeted sensors, largely due to a more alkaline local pH environment. These observations support the view that mitochondrial glutathione redox state is maintained and regulated independently from that of the cytosol and nucleus. Taken together, our data show the robustness of the rxYFP sensors to measure compartmental redox changes in human cells. Complementary to existing redox sensors and conventional redox measurements, compartment-targeted rxYFP sensors provide a novel tool for examining mammalian cell redox homeostasis, permitting high-resolution readout of steady glutathione state and dynamics of redox changes.

Peroxiredoxin 1 knockdown potentiates β-lapachone cytotoxicity through modulation of reactive oxygen species and mitogen-activated protein kinase signals

Peroxiredoxin (Prx) 1 is a member of the thiol-specific peroxidases family and plays diverse roles such as H2O2 scavenger, redox signal transducer and molecular chaperone. Prx1 has been reported to be involved in protecting cancer cells against various therapeutic challenges. We investigated how modulations of intracellular redox system affect cancer cell sensitivity to reactive oxygen species (ROS)-generating drugs. We observed that stable and transient Prx1 knockdown significantly enhanced HeLa cell sensitivity to β-lapachone (β-lap), a potential anticancer agent. Prx1 knockdown markedly potentiated 2 µM β-lap-induced cytotoxicity through ROS accumulation. This effect was largely NAD(P)H:quinone oxidoreductase 1 dependent and associated with a decrease in poly(ADP-ribose) polymerase 1 protein levels, phosphorylation of JNK, p38 and Erk proteins in mitogen-activated protein kinase (MAPK) pathways and a decrease in thioredoxin 1 (Trx1) protein levels. Trx1 serves as an electron donor for Prx1 and is overexpressed in Prx1 knockdown cells. Based on the fact that Prx1 is a major ROS scavenger and a partner of at least ASK1 and JNK, two key components of MAPK pathways, we propose that Prx1 knockdown-induced sensitization to β-lap is achieved through combined action of accumulation of ROS and enhancement of MAPK pathway activation, leading to cell apoptosis. These data support the view that modulation of intracellular redox state could be an alternative approach to enhance cancer cell sensitivity to ROS-generating drugs or to overcome some types of drug resistance.

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

SummaryThe RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.

Mismatch-stimulated plasmid integration in yeast.

SummaryA single base pair mismatch (G:T or A:C) in the CYC1 gene of the integrative plasmid pAB218 stimulates up to a five-fold integration into the yeast chromosome. Analysis of chromosomal sites of plasmid integration suggests that the mismatch-stimulated integration is not targeted as would be expected if crossovers, localised in the region of the mismatch, were a necessary step in mismatch repair. Instead, the observed mismatch-stimulated plasmid integration could be due to potentially recombinogenic structures formed during mismatch repair, such as single-stranded gaps or denatured DNA regions extending around the plasmid molecule.