High levels of transcription in Saccharomyces cerevisiae are associated with increased genetic instability, which has been linked to DNA damage. Here, we describe a pGAL-CAN1 forward mutation assay for studying transcription-associated mutagenesis (TAM) in yeast. In a wild-type background with no alterations in DNA repair capacity, ≈50% of forward mutations that arise in the CAN1 gene under high-transcription conditions are deletions of 2-5 bp. Furthermore, the deletions characteristic of TAM localize to discrete hotspots that coincide with 2-4 copies of a tandem repeat. Although the signature deletions of TAM are not affected by the loss of error-free or error-prone lesion bypass pathways, they are completely eliminated by deletion of the TOP1 gene, which encodes the yeast type IB topoisomerase. Hotspots can be transposed into the context of a frameshift reversion assay, which is sensitive enough to detect Top1-dependent deletions even in the absence of high transcription. We suggest that the accumulation of Top1 cleavage complexes is related to the level of transcription and that their removal leads to the signature deletions. Given the high degree of conservation between DNA metabolic processes, the links established here among transcription, Top1, and mutagenesis are likely to extend beyond the yeast system.

The RAD21/REC8 gene family has been implicated in sister chromatid cohesion and DNA repair in several organisms. Unlike most eukaryotes, Arabidopsis thaliana has three RAD21 gene homologues, and their cloning and characterization are reported here. All three genes, AtRAD21.1, AtRAD21.2, and AtRAD21.3, are expressed in tissues rich in cells undergoing cell division, and AtRAD21.3 shows the highest relative level of expression. An increase in steady-state levels of AtRAD21.1 transcript was also observed, specifically after the induction of DNA damage. Phenotypic analysis of the atrad21.1 and atrad21.3 mutants revealed that neither of the single mutants was lethal, probably due to the redundancy in function of the AtRAD21 genes. However, AtRAD21.1 plays a critical role in recovery from DNA damage during seed imbibition, prior to germination, as atrad21.1 mutant seeds are hypersensitive to radiation damage.

A shift in toxicity testing from in vivo to in vitro may efficiently prioritize compounds, reveal new mechanisms, and enable predictive modeling. Quantitative high-throughput screening (qHTS) is a major source of data for computational toxicology, and our goal in this study was to aid in the development of predictive in vitro models of chemical-induced toxicity, anchored on interindividual genetic variability. Eighty-one human lymphoblast cell lines from 27 Centre d'Etude du Polymorphisme Humain trios were exposed to 240 chemical substances (12 concentrations, 0.26nM-46.0μM) and evaluated for cytotoxicity and apoptosis. qHTS screening in the genetically defined population produced robust and reproducible results, which allowed for cross-compound, cross-assay, and cross-individual comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, whereas others exhibited interindividual differences in cytotoxicity. Specifically, the qHTS in a population-based human in vitro model system has several unique aspects that are of utility for toxicity testing, chemical prioritization, and high-throughput risk assessment. First, standardized and high-quality concentration-response profiling, with reproducibility confirmed by comparison with previous experiments, enables prioritization of chemicals for variability in interindividual range in cytotoxicity. Second, genome-wide association analysis of cytotoxicity phenotypes allows exploration of the potential genetic determinants of interindividual variability in toxicity. Furthermore, highly significant associations identified through the analysis of population-level correlations between basal gene expression variability and chemical-induced toxicity suggest plausible mode of action hypotheses for follow-up analyses. We conclude that as the improved resolution of genetic profiling can now be matched with high-quality in vitro screening data, the evaluation of the toxicity pathways and the effects of genetic diversity are now feasible through the use of human lymphoblast cell lines.

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