[Relationship between Type I and Type II Template Processes: Amyloids and Genome Stability].

Classical views of hereditary mechanisms consider linear nucleic acids, DNA and RNA, as template molecules wherein genetic information is encoded by the sequence of nitrogenous bases. The template principle embodied in the central dogma of molecular biology describes the allowed paths of genetic information transfer from nucleic acids to proteins. The discovery of prions revealed an additional hereditary mechanism whereby the spatial structure is transmitted from one protein molecule to another independently of the sequence of nitrogenous bases in their structural genes. The simultaneous existence of linear (type I) and conformational (type II) templates in one cell inevitably implies their interaction. The review analyzes the current data confirming the idea that protein amyloid transformation may influence the genome stability and considers potential mechanisms of interactions between type I and type II template processes. Special attention is paid to the joint contribution of the two process to tumor "evolution" and the mechanisms of genome destabilization due to amyloid transformation of proteins in Alzheimer's and Parkinson's diseases and Down syndrome.

Deletion of the DEF1 gene does not confer UV-immutability but frequently leads to self-diploidization in yeast Saccharomyces cerevisiae.

In yeast Saccharomyces cerevisiae, the DEF1 gene is responsible for regulation of many cellular processes including ubiquitin-dependent degradation of DNA metabolism proteins. Recently it has been proposed that Def1 promotes degradation of the catalytic subunit of DNA polymerase δ at sites of DNA damage and regulates a switch to specialized polymerases and, as a consequence, DNA-damage induced mutagenesis. The idea was based substantially on the severe defects in induced mutagenesis observed in the def1 mutants. We describe that UV mutability of def1Δ strains is actually only moderately affected, while the virtual absence of UV mutagenesis in many def1Δ clones is caused by a novel phenotype of the def1 mutants, proneness to self-diploidization. Diploids are extremely frequent (90%) after transformation of wild-type haploids with def1::kanMX disruption cassette and are frequent (2.3%) in vegetative haploid def1 cultures. Such diploids look "UV immutable" when assayed for recessive forward mutations but have normal UV mutability when assayed for dominant reverse mutations. The propensity for frequent self-diploidization in def1Δ mutants should be taken into account in studies of the def1Δ effect on mutagenesis. The true haploids with def1Δ mutation are moderately UV sensitive but retain substantial UV mutagenesis for forward mutations: they are fully proficient at lower doses and only partially defective at higher doses of UV. We conclude that Def1 does not play a critical role in damage-induced mutagenesis.

Defect of Fe-S cluster binding by DNA polymerase δ in yeast suppresses UV-induced mutagenesis, but enhances DNA polymerase ζ - dependent spontaneous mutagenesis.

Eukaryotic genomes are duplicated by a complex machinery, utilizing high fidelity replicative B-family DNA polymerases (pols) α, δ and ε. Specialized error-prone pol ζ, the fourth B-family member, is recruited when DNA synthesis by the accurate trio is impeded by replication stress or DNA damage. The damage tolerance mechanism dependent on pol ζ prevents DNA/genome instability and cell death at the expense of increased mutation rates. The pol switches occurring during this specialized replication are not fully understood. The loss of pol ζ results in the absence of induced mutagenesis and suppression of spontaneous mutagenesis. Disruption of the Fe-S cluster motif that abolish the interaction of the C-terminal domain (CTD) of the catalytic subunit of pol ζ with its accessory subunits, which are shared with pol δ, leads to a similar defect in induced mutagenesis. Intriguingly, the pol3-13 mutation that affects the Fe-S cluster in the CTD of the catalytic subunit of pol δ also leads to defective induced mutagenesis, suggesting the possibility that Fe-S clusters are essential for the pol switches during replication of damaged DNA. We confirmed that yeast strains with the pol3-13 mutation are UV-sensitive and defective in UV-induced mutagenesis. However, they have increased spontaneous mutation rates. We found that this increase is dependent on functional pol ζ. In the pol3-13 mutant strain with defective pol δ, there is a sharp increase in transversions and complex mutations, which require functional pol ζ, and an increase in the occurrence of large deletions, whose size is controlled by pol ζ. Therefore, the pol3-13 mutation abrogates pol ζ-dependent induced mutagenesis, but allows for pol ζ recruitment for the generation of spontaneous mutations and prevention of larger deletions. These results reveal differential control of the two major types of pol ζ-dependent mutagenesis by the Fe-S cluster present in replicative pol δ.

EVALUATION OF EFFECTIVENESS OF SYNCHRONIZATION METHODS OF CELL DIVISION IN YEAST SACCHAROMYCES CEREVISIAE.

Synchronization of cell division in cultures of yeast Saccharomyces cerevisiae is widely used in research on the regulation of gene expression and biochemical processes in eukaryotes at different stages of the cell cycle. Here, we compare the efficiency of modern most commonly used methods to achieve and assess the degree of synchronization of cell division in yeast. Block-and-release methods with alpha-factor, hydroxyurea, nocodazole, cdc28-4 mutation are described in detail with practical notes.

Participation of translesion synthesis DNA polymerases in the maintenance of chromosome integrity in yeast Saccharomyces cerevisiae.

We employed a genetic assay based on illegitimate hybridization of heterothallic Saccharomyces cerevisiae strains (the α-test) to analyze the consequences for genome stability of inactivating translesion synthesis (TLS) DNA polymerases. The α-test is the only assay that measures the frequency of different types of mutational changes (point mutations, recombination, chromosome or chromosome arm loss) and temporary changes in genetic material simultaneously. All these events are manifested as illegitimate hybridization and can be distinguished by genetic analysis of the hybrids and cytoductants. We studied the effect of Polζ, Polη, and Rev1 deficiency on the genome stability in the absence of genotoxic treatment and in UV-irradiated cells. We show that, in spite of the increased percent of accurately repaired primary lesions, chromosome fragility, rearrangements, and loss occur in the absence of Polζ and Polη. Our findings contribute to further refinement of the current models of translesion synthesis and the organization of eukaryotic replication fork.

[The role of metabolic activation of promutagens in the genome destabilization under pheromonal stress in the house mouse (Mus musculus)].

The hypothesis on a relationship between the high frequency of mitotic disturbances in bone marrow cells and the change in the activity of the S9 liver fraction containing promutagen-activating enzymes under olfactory stress in the house mouse Mus musculus has been tested. For this purpose, the effect of the pheromone 2,5-dimethylpyrazine on the frequency of mitotic disturbances in mouse bone marrow cells has been measured by the anaphase-telophase assay. The Ames test using Salmonella typhimurium has been employed to compare the capacities of the S9 liver fractions from stressed and intact mice for activating the promutagen 2-aminofluorene. It has been demonstrated that the increased frequency of mitotic disturbances in bone marrow cells induced by the pheromonal stressor in male house mice is accompanied by an increased promutagen-activating capacity of the S9 liver fraction. The model system used in the study allowed the genetic consequences of the exposure to the olfactory stressor to be estimated and the possible mechanisms of genome destabilization to be assumed.

[Phenotypic manifestation and trans-conversion of primary genetic material damages considered in the alpha-test on the yeast Saccharomyces cerevisiae].

Primary (spontaneous and externally induces) damages to genetic material frequently lead to heritable changes (gene mutations, chromosome aberrations and nondisjunction), which may cause cancer inherent and inborn diseases. It is suggested that primary damages may affect a phenotype until they are repaired or become mutations during inaccurate repair The alpha-test on the yeast Saccharomyces cerevisiae can answer the fundamental questions as the nature of primary damages that can be phenotypically manifested, their occurrence, conversion to each other and repair or conversion to heritable changes in genetic material.

[Genetic control of metabolism of mutagenic purine base analogs 6-hydroxylaminopurine and 2-amino-6-hydroxylaminopurine in yeast Saccharomyces cerevisiae].

The influence of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP) de novo and the purine utilization and interconversion pathway, on sensitivity of yeast Saccharomyces cerevisiae cells to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA) was studied. It was shown that the manifestation of HAP and AHA mutagenic properties involves the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of each stage of IMP biosynthesis, with the exception of the block mediated by inactivation of genes ADE16 and ADE17 leading to the accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), was shown to enhance yeast cell sensitivity to the HAP mutagenic effect; however, it does not affect the sensitivity to AHA. A blockade of conversion of IMP into adenosine monophosphate (AMP) causes hypersensitivity of yeast cells to the mutagenic action of HAP and to the toxic effect of HAP, AHA, and hypoxanthine. It is fully probable that this enhancement of sensitivity to HAP and AHA is conditioned by changes in the pool of purines. This indicates that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine utilization and interconversion in yeast make the greatest contribution to the system of protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are considered.