Hanan Al-Refai

Most errors that arise during DNA replication can be corrected by DNA polymerase proofreading or by post-replication mismatch repair (MMR). Inactivation of both mutation-avoidance systems results in extremely high mutability that can lead to error catastrophe. High mutability and the likelihood of cancer can be caused by mutations and epigenetic changes that reduce MMR. Hypermutability can also be caused by external factors that directly inhibit MMR. Identifying such factors has important implications for understanding the role of the environment in genome stability. We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen, can result in extreme hypermutability. The mutation specificity along with responses in proofreading-deficient and MMR-deficient mutants indicate that cadmium reduces the capacity for MMR of small misalignments and base-base mismatches. In extracts of human cells, cadmium inhibited at least one step leading to mismatch removal. Together, our data show that a high level of genetic instability can result from environmental impediment of a mutation-avoidance system.

A variety of pathologies are associated with exposure to supraphysiological concentrations of essential metals and to non-essential metals and metalloids. The molecular mechanisms linking metal exposure to human pathologies have not been clearly defined. To address these gaps in our understanding of the molecular biology of transition metals, the genomic effects of exposure to Group IB (copper, silver), IIB (zinc, cadmium, mercury), VIA (chromium), and VB (arsenic) elements on the yeast Saccharomyces cerevisiae were examined. Two comprehensive sets of metal-responsive genomic profiles were generated following exposure to equi-toxic concentrations of metal: one that provides information on the transcriptional changes associated with metal exposure (transcriptome), and a second that provides information on the relationship between the expression of ∼4,700 non-essential genes and sensitivity to metal exposure (deletome). Approximately 22% of the genome was affected by exposure to at least one metal. Principal component and cluster analyses suggest that the chemical properties of the metal are major determinants in defining the expression profile. Furthermore, cells may have developed common or convergent regulatory mechanisms to accommodate metal exposure. The transcriptome and deletome had 22 genes in common, however, comparison between Gene Ontology biological processes for the two gene sets revealed that metal stress adaptation and detoxification categories were commonly enriched. Analysis of the transcriptome and deletome identified several evolutionarily conserved, signal transduction pathways that may be involved in regulating the responses to metal exposure. In this study, we identified genes and cognate signaling pathways that respond to exposure to essential and non-essential metals. In addition, genes that are essential for survival in the presence of these metals were identified. This information will contribute to our understanding of the molecular mechanism by which organisms respond to metal stress, and could lead to an understanding of the connection between environmental stress and signal transduction pathways.

Until recently, the only biological function attributed to the 3'-->5' exonuclease activity of DNA polymerases was proofreading of replication errors. Based on genetic and biochemical analysis of the 3'-->5' exonuclease of yeast DNA polymerase delta (Pol delta) we have discerned additional biological roles for this exonuclease in Okazaki fragment maturation and mismatch repair. We asked whether Pol delta exonuclease performs all these biological functions in association with the replicative complex or as an exonuclease separate from the replicating holoenzyme. We have identified yeast Pol delta mutants at Leu523 that are defective in processive DNA synthesis when the rate of misincorporation is high because of a deoxynucleoside triphosphate (dNTP) imbalance. Yet the mutants retain robust 3'-->5' exonuclease activity. Based on biochemical studies, the mutant enzymes appear to be impaired in switching of the nascent 3' end between the polymerase and the exonuclease sites, resulting in severely impaired biological functions. Mutation rates and spectra and synergistic interactions of the pol3-L523X mutations with msh2, exo1, and rad27/fen1 defects were indistinguishable from those observed with previously studied exonuclease-defective mutants of the Pol delta. We conclude that the three biological functions of the 3'-->5' exonuclease addressed in this study are performed intramolecularly within the replicating holoenzyme.