Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Comparative Genomic Screen in Two Yeasts Reveals Conserved Pathways in the Response Network to Phenol Stress.

Living organisms encounter various perturbations, and response mechanisms to such perturbations are vital for species survival. Defective stress responses are implicated in many human diseases including cancer and neurodegenerative disorders. Phenol derivatives, naturally occurring and synthetic, display beneficial as well as detrimental effects. The phenol derivatives in this study, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and bisphenol A (BPA), are widely used as food preservatives and industrial chemicals. Conflicting results have been reported regarding their biological activity and correlation with disease development; understanding the molecular basis of phenol action is a key step for addressing issues relevant to human health. This work presents the first comparative genomic analysis of the genetic networks for phenol stress response in an evolutionary context of two divergent yeasts, Schizosaccharomyces pombe and Saccharomyces cerevisiae Genomic screening of deletion strain libraries of the two yeasts identified genes required for cellular response to phenol stress, which are enriched in human orthologs. Functional analysis of these genes uncovered the major signaling pathways involved. The results provide a global view of the biological events constituting the defense process, including cell cycle arrest, DNA repair, phenol detoxification by V-ATPases, reactive oxygen species alleviation, and endoplasmic reticulum stress relief through ergosterol and the unfolded protein response, revealing novel roles for these cellular pathways.

Growth Inhibition and DNA Damage Induced by X-Phenols in Yeast: A Quantitative Structure-Activity Relationship Study.

Phenolic compounds and their derivatives are ubiquitous constituents of numerous synthetic and natural chemicals that exist in the environment. Their toxicity is mostly attributed to their hydrophobicity and/or the formation of free radicals. In a continuation of the study of phenolic toxicity in a systematic manner, we have examined the biological responses of Saccharomyces cerevisiae to a series of mostly monosubstituted phenols utilizing a quantitative structure-activity relationship (QSAR) approach. The biological end points included a growth assay that determines the levels of growth inhibition induced by the phenols as well as a yeast deletion (DEL) assay that assesses the ability of X-phenols to induce DNA damage or DNA breaks. The QSAR analysis of cell growth patterns determined by IC50 and IC80 values indicates that toxicity is delineated by a hydrophobic, parabolic model. The DEL assay was then utilized to detect genomic deletions in yeast. The increase in the genotoxicity was enhanced by the electrophilicity of the phenolic substituents that were strong electron donors as well as by minimal hydrophobicity. The electrophilicities are represented by Brown's sigma plus values that are a variant of the Hammett sigma constants. A few mutant strains of genes involved in DNA repair were separately exposed to 2,6-di-tert-butyl-4-methyl-phenol (BHT) and butylated hydroxy anisole (BHA). They were subsequently screened for growth phenotypes. BHA-induced growth defects in most of the DNA repair null mutant strains, whereas BHT was unresponsive.