N. A. Koltovaya

Mutations of the CDC28 Gene and the Radiation Sensitivity of Saccharomyces cerevisiae

CDC28‐srm, a non‐temperature‐sensitive (ts) mutation in the CDC28 gene of Saccharomyces cerevisiae that affects fidelity of mitotic transmission of both mitochondrial and nuclear genetic structures (Devin et al., 1990), also affected cell growth and sensitivity to lethal effects of ionizing radiation. At 30°C CDC28‐13, a ts mutation, was without appreciable effects on spontaneous mitochondrial rho−‐mutagenesis, cell growth and radiation sensitivity, whereas all three cell characteristics mentioned were affected (although to a lesser degree than by CDC28‐srm) by CDC28‐1, another ts mutation. CDC28‐srm was without any significant effect on the rates of spontaneous nuclear gene mutations and γ‐ray‐induced mitotic recombination. An analysis of double mutants as regards their radiation sensitivity has revealed additive or even synergistic interactions between the CDC28‐srm mutation and every one of the rad6‐1 and rad52‐1 mutations. The rad9Δ allele was found to be epistatic to CDC28‐srm. These data suggest that the p34CDC28 protein is involved in the RAD9‐dependent feedback control of DNA integrity operating at the cell cycle checkpoints.

News & Notes: Correlation of Resistance to the Alkaloid Lycorine with the Degree of Suppressiveness in Petite Mutants of Saccharomyces cerevisiae

Abstract. In previous papers (Del Giudice et al. Curr Genet 8:493–497, 1984; Massardo et al. Curr Genet 17:455–457, 1985) we have shown that strains of Saccharomyces cerevisiae that are devoid of mitochondrial DNA (rhoo) are resistant to the alkaloid lycorine isolated from Amaryllis plants, whereas strains containing mitochondrial DNA (rho−, mit−, or rho+) are sensitive to this drug. In addition, we were able to show that the so-called hypersuppressive petites, whose mitochondrial genomes consist of short regions of DNA containing an ori sequence, show intermediate resistance. In this paper, we demonstrate that the degree of suppressiveness of a rho− mutant correlates with the degree of resistance to lycorine.

NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis

An increase in the mitochondrial rho− mutagenesis is a well‐known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho− mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell‐cycle regulation gene, result in a decrease in spontaneous rho− mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein–protein interactions in the yeast cell. The effects of four mutations, including net1‐srm and hfi1‐srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cells sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast. Copyright

Activation of repair and checkpoints by double-strand DNA breaks: Activational cascade of protein phosphorylation

Molecular mechanisms of activation of repair and checkpoints by DNA double-strand breaks are considered. They include phosphorylation by protein kinases of repair and checkpoint proteins resulting in their activation, alteration of affinity to other proteins, and alteration of their localization.

[Involvement of cyclin-dependent kinase CDK1/CDC28 in regulation of cell cycle].

Cyclin-dependent kinases (CDKs) are a family of enzymes essential for the progression of the cells through the cell cycle in eukaryotes. Moreover, genetic stability-maintaining processes, such as check-point control and DNA repair, require the phosphorylation of a wide variety of target substrates by CDK. In budding yeast Saccharomyces cerevisiae, the key role in the cell cycle progression is played by CDK1/CDC28 kinase. This enzyme is the most thoroughly investigated. In this review the involvement of CDC28 kinase in regulation of the cell cycle is discussed in the light of newly obtained data.

[Interaction between checkpoint genes RAD9, RAD17, RAD24, and RAD53 involved in the determination of yeast Saccharomyces cerevisiae sensitivity to ionizing radiation].

Mechanisms for genetic control of cell division cycle (checkpoint control) have been studied in most detail in yeast Saccharomyces cerevisiae. To clarify the role of checkpoint genes RAD9, RAD17, RAD24, and RAD53 in cell radioresistance, double mutants were analyzed for cell sensitivity to ionizing radiation. Double mutants carrying mutations in combination with mutation rad9Δ were shown to manifest the epistatic type of interaction. Our results suggest that checkpoint genes RAD9, RAD17, RAD24, and RAD53 belong to a single epistatic group designated RAD9 and govern the same pathway. Genes RAD9 and RAD53 have a positive effect on sensitivity to γ-radiation, whereas RAD17 and RAD24 have a negative effect. Interactions between mutations may differ when considering their sensitivity to γ-radiation and UV light; mutations rad9Δ and rad24Δ were shown to manifest the additive effect in the first case and epistatic effect in the second.

[Participation of SRM5/CDC28, SRM8/NET1, and SRM12/HFI1 genes in checkpoint control in yeast Saccharomyces cerevisiae].

About twenty genes participating in checkpoint control are known in yeast Saccharomyces cerevisiae. The involvement of SRM genes in the cell cycle arrest under the action of DNA damaging agents was studied in this work. These genes were earlier defined as genes affecting genetic stability and radiosensitivity. It was shown that mutations srm5/cdc28-srm, srm8/net1-srm, and srm12/hfi1-srm fail the cell cycle arrest in the presence of DNA damage and influence the checkpoint arrest in G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm5, srm12), and G2/M (srm5). It seems likely that genes SRM5/CDC28, SRM12/HFI1/ADA1, and SRM8/NET1 are involved in a cell response to DNA damage, and in checkpoint regulation in particular.

Interaction between checkpoint genes RAD9, RAD17, RAD24, RAD53, and genes SRM5/CDC28, SRM8/NET1, and SRM12/HFI1 involved in the determination of yeast Saccharomyces cerevisiae sensitivity to ionizing radiation

Analysis of radiosensitivity of double mutants in the yeast Saccharomyces cerevisiae revealed that checkpoint genes RAD9, RAD17, RAD24, and RAD53 along with genes CDC28 and NET1 belong to one epistasis group designated the RAD9 group. The use of srm mutations allowed the demonstration of a branched RAD9-dependent pathway of cell radioresistance. Mutation cdc28-srm is hypostatic to rad9Δ, rad17Δ, and rad24Δ being additive with rad53. Mutation net1-srm is hypostatic to rad9Δ and rad53 but additively enhance the effects of mutations rad17Δ and rad24Δ. Gene SRM12/HFI1 is not a member of the RAD9 group. Mutation in gene hfi1-srm manifests the additive effect on mutations rad24Δ and rad9Δ. The analyzed genes can also participate in minor mechanisms of radioresistance that are relatively independent of the above RAD9-dependent mechanism.

New Mutations of SRMGenes in Saccharomyces cerevisiae and Certain Characteristics of Their Phenotypic Effects

The effects of the previously identified mutations in nuclear genes SRM8, SRM12, SRM15, and SRM17on the maintenance of chromosomes and recombinant plasmids in Saccharomyces cerevisiaecells and on cell sensitivity to ionizing radiation were studied. The srm8mutation caused an increase in spontaneous chromosome loss in diploid cells. In yeast cells with the intact mitochondrial genome, all examined srmmutations decreased the mitotic stability of a centromeric recombinant plasmid with the chromosomal ARS element. Mutations srm12, srm15, and srm17also decreased the mitotic stability of a centromereless plasmid containing the same ARS element, whereas the srm8mutation did not markedly affect the maintenance of this plasmid. Mutations srm8, srm12, and srm17were shown to increase cell sensitivity to γ-rays. The SRM8gene was mapped, cloned, and found to correspond to the open reading frame YJLO76w in chromosome X.

Kinase Cascade of DNA Damage Checkpoint

Molecular mechanisms of activation of DNA repair and checkpoint by double-strand breaks are considered. They include phosphorylation by protein kinases of repair and checkpoint proteins resulting in their activation, alteration of affinity to other proteins, and changes of their localization.