CRISPR/Cas9 as a Mutagenic Factor.

The discovery of the CRISPR/Cas9 microbial adaptive immune system has revolutionized the field of genetics, by greatly enhancing the capacity for genome editing. CRISPR/Cas9-based editing starts with DNA breaks (or other lesions) predominantly at target sites and, unfortunately, at off-target genome sites. DNA repair systems differing in accuracy participate in establishing desired genetic changes but also introduce unwanted mutations, that may lead to hereditary, oncological, and other diseases.

Detection of Primary DNA Lesions by Transient Changes in Mating Behavior in Yeast Using the Alpha-Test.

Spontaneous or induced DNA lesions can result in stable gene mutations and chromosomal aberrations due to their inaccurate repair, ultimately resulting in phenotype changes. Some DNA lesions per se may interfere with transcription, leading to temporary phenocopies of mutations. The direct impact of primary DNA lesions on phenotype before their removal by repair is not well understood.

Interaction of Prions Causes Heritable Traits in Saccharomyces cerevisiae.

The concept of "protein-based inheritance" defines prions as epigenetic determinants that cause several heritable traits in eukaryotic microorganisms, such as Saccharomyces cerevisiae and Podospora anserina. Previously, we discovered a non-chromosomal factor, [NSI+], which possesses the main features of yeast prions, including cytoplasmic infectivity, reversible curability, dominance, and non-Mendelian inheritance in meiosis. This factor causes omnipotent suppression of nonsense mutations in strains of S.

Prions, amyloids, and RNA: Pieces of a puzzle.

Amyloids are protein aggregates consisting of fibrils rich in β-sheets. Growth of amyloid fibrils occurs by the addition of protein molecules to the tip of an aggregate with a concurrent change of a conformation. Thus, amyloids are self-propagating protein conformations.

Genome Sequencing and Comparative Analysis of Saccharomyces cerevisiae Strains of the Peterhof Genetic Collection.

The Peterhof genetic collection of Saccharomyces cerevisiae strains (PGC) is a large laboratory stock that has accumulated several thousands of strains for over than half a century. It originated independently of other common laboratory stocks from a distillery lineage (race XII). Several PGC strains have been extensively used in certain fields of yeast research but their genomes have not been thoroughly explored yet.

Modulation of efficiency of translation termination in Saccharomyces cerevisiae.

Nonsense suppression is a readthrough of premature termination codons. It typically occurs either due to the recognition of stop codons by tRNAs with mutant anticodons, or due to a decrease in the fidelity of translation termination. In the latter case, suppressors usually promote the readthrough of different types of nonsense codons and are thus called omnipotent nonsense suppressors.

[NSI+] determinant has a pleiotropic phenotypic manifestation that is modulated by SUP35, SUP45, and VTS1 genes.

We recently discovered the novel non-chromosomal determinant in Saccharomyces cerevisiae [NSI(+)] (nonsense suppression inducer), which causes omnipotent nonsense suppression in strains where the Sup35 N-terminal domain is deleted. [NSI(+)] possesses yeast prion features and does not correspond to previously identified yeast prion determinants. Here, we show that [NSI(+)] enhances nonsense codon read-through and inhibits vegetative growth in S.

[NSI (+)]: a novel non-Mendelian nonsense suppressor determinant in Saccharomyces cerevisiae.

Non-Mendelian determinants that control heritable traits in yeast are subdivided into two major groups-one that includes DNA- or RNA-based elements and another that comprises protein-based factors that are analogous to mammalian prion. All yeast non-Mendelian determinants show dominant inheritance, and some of them demonstrate cytoplasmic infectivity. Only prions, however, harbor-specific features, such as high frequency of induction following overproduction of prion-encoding protein, loss of the protein's normal function, and reversible curability.

Biological roles of prion domains.

In vivo amyloid formation is a widespread phenomenon in eukaryotes. Self-perpetuating amyloids provide a basis for the infectious or heritable protein isoforms (prions). At least for some proteins, amyloid-forming potential is conserved in evolution despite divergence of the amino acid (aa) sequences.

Prion-dependent lethality of sup45 mutants in Saccharomyces cerevisiae.

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI(+)]. It is known that combination of [PSI(+)] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality.

N-terminal extension of Saccharomyces cerevisiae translation termination factor eRF3 influences the suppression efficiency of sup35 mutations.

The eukaryotic translation termination factor eRF3 stimulates release of nascent polypeptides from the ribosome in a GTP-dependent manner. In most eukaryotes studied, eRF3 consists of an essential, conserved C-terminal domain and a nonessential, nonconserved N-terminal extension. However, in some species, this extension is required for efficient termination.

Prion variant maintained only at high levels of the Hsp104 disaggregase.

The yeast prion [PSI(+)] is a self-perpetuating aggregated isoform of the translation termination factor Sup35. [PSI ( + )] propagation is promoted by moderate levels and antagonized by high levels of the chaperone Hsp104. In agreement with the model postulating that excess Hsp104 acts on [PSI ( + )] by disaggregating prion polymers, we show that an increase in Sup35 levels, accompanied by an increase in size of prion aggregates, also partially protects [PSI(+)] from elimination by excess Hsp104.

Poly(A)-binding protein acts in translation termination via eukaryotic release factor 3 interaction and does not influence [PSI(+)] propagation.

Recent studies of translational control suggest that translation termination may not be simply the end of synthesizing a protein but rather be involved in modulating both the translation efficiency and stability of a given transcript. Using recombinant eukaryotic release factor 3 (eRF3) and cellular extracts, we have shown for Saccharomyces cerevisiae that yeast eRF3 and Pab1p can interact. This interaction, mediated by the N+M domain of eRF3 and amino acids 473 to 577 of Pab1p, was demonstrated to be direct by the two-hybrid approach.

Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae.

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI(+)], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN(+)], determines the probability of de novo generation of [PSI(+)] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p.