Nonmutagenic carcinogens induce intrachromosomal recombination in dividing yeast cells.

A large number of animal and human carcinogens without apparent genotoxic activity exist (nonmutagenic carcinogens) that are difficult or impossible to detect with the currently used short-term tests. Because of the association of carcinogenesis with genome rearrangement, a system selecting for intrachromosomal recombination (DEL recombination) that results in genome rearrangement has been constructed in the yeast Saccharomyces cerevisiae. Because DEL recombination is under different genetic control than interchromosomal recombination and meiotic recombination, it is probably due to a different mechanism. It has been found that DEL recombination is readily inducible by 10 mutagenic carcinogens and 17 nonmutagenic carcinogens that are not detectable (false negatives) with the Ames assay. In addition, three out of four mutagens that do not cause cancer (false positives in the Ames assay) do not induce DEL recombination. DEL recombination is inducible by UV only in dividing cells but not in cells synchronized in the G1 or G2 phase of the cell cycle. Interchromosomal recombination, on the other hand, is inducible in G1 but not in G2. The nonmutagenic carcinogens induce DEL recombination only in actively growing cells, which may give some indication as to their mechanism. Further characterization of the mechanism involved in induction of DEL recombination may contribute to the understanding of the biological activity of nonmutagenic carcinogens

Srs2 mediates PCNA-SUMO-dependent inhibition of DNA repair synthesis

Completion of DNA replication needs to be ensured even when challenged with fork progression problems or DNA damage. PCNA and its modifications constitute a molecular switch to control distinct repair pathways. In yeast, SUMOylated PCNA (S-PCNA) recruits Srs2 to sites of replication where Srs2 can disrupt Rad51 filaments and prevent homologous recombination (HR). We report here an unexpected additional mechanism by which S-PCNA and Srs2 block the synthesis-dependent extension of a recombination intermediate, thus limiting its potentially hazardous resolution in association with a cross-over. This new Srs2 activity requires the SUMO interaction motif at its C-terminus, but neither its translocase activity nor its interaction with Rad51. Srs2 binding to S-PCNA dissociates Polδ and Polη from the repair synthesis machinery, thus revealing a novel regulatory mechanism controlling spontaneous genome rearrangements. Our results suggest that cycling cells use the Siz1-dependent SUMOylation of PCNA to limit the extension of repair synthesis during template switch or HR and attenuate reciprocal DNA strand exchanges to maintain genome stability. © 2013 European Molecular Biology Organization

Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence

Increased retention of functional fusions to toxic genes in new two-hybrid libraries of the E. coli strain MG1655 and B. subtilis strain 168 genomes, prepared without passaging through E. coli

BACKGROUND: Cloning of genes in expression libraries, such as the yeast two-hybrid system (Y2H), is based on the assumption that the loss of target genes is minimal, or at worst, managable. However, the expression of genes or gene fragments that are capable of interacting with E. coli or yeast gene products in these systems has been shown to be growth inhibitory, and therefore these clones are underrepresented (or completely lost) in the amplified library. RESULTS: Analysis of candidate genes as Y2H fusion constructs has shown that, while stable in E. coli and yeast for genetic studies, they are rapidly lost in growth conditions for genomic libraries. This includes the rapid loss of a fragment of the E. coli cell division gene ftsZ which encodes the binding site for ZipA and FtsA. Expression of this clone causes slower growth in E. coli. This clone is also rapidly lost in yeast, when expressed from a GAL1 promoter, relative to a vector control, but is stable when the promoter is repressed. We have demonstrated in this report that the construction of libraries for the E. coli and B. subtilis genomes without passaging through E. coli is practical, but the number of transformants is less than for libraries cloned using E. coli as a host. Analysis of several clones in the libraries that are strongly growth inhibitory in E. coli include genes for many essential cellular processes, such as transcription, translation, cell division, and transport. CONCLUSION: Expression of Y2H clones capable of interacting with E. coli and yeast targets are rapidly lost, causing a loss of complexity. The strategy for preparing Y2H libraries described here allows the retention of genes that are toxic when inappropriately expressed in E. coli, or yeast, including many genes that represent potential antibacterial targets. While these methods are generally applicable to the generation of Y2H libraries from any source, including mammalian and plant genomes, the potential of functional clones interacting with host proteins to inhibit growth would make this approach most relevant for the study of prokaryotic genomes

RAD59 and RAD1 cooperate in translocation formation by single-strand annealing in Saccharomyces cerevisiae

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails

Msh2 Blocks an Alternative Mechanism for Non-Homologous Tail Removal during Single-Strand Annealing in Saccharomyces cerevisiae

Chromosomal translocations are frequently observed in cells exposed to agents that cause DNA double-strand breaks (DSBs), such as ionizing radiation and chemotherapeutic drugs, and are often associated with tumors in mammals. Recently, translocation formation in the budding yeast, Saccharomyces cerevisiae, has been found to occur at high frequencies following the creation of multiple DSBs adjacent to repetitive sequences on non-homologous chromosomes. The genetic control of translocation formation and the chromosome complements of the clones that contain translocations suggest that translocation formation occurs by single-strand annealing (SSA). Among the factors important for translocation formation by SSA is the central mismatch repair (MMR) and homologous recombination (HR) factor, Msh2. Here we describe the effects of several msh2 missense mutations on translocation formation that suggest that Msh2 has separable functions in stabilizing annealed single strands, and removing non-homologous sequences from their ends. Additionally, interactions between the msh2 alleles and a null allele of RAD1, which encodes a subunit of a nuclease critical for the removal of non-homologous tails suggest that Msh2 blocks an alternative mechanism for removing these sequences. These results suggest that Msh2 plays multiple roles in the formation of chromosomal translocations following acute levels of DNA damage

High-Throughput Screen for Identifying Small Molecules That Target Fungal Zinc Homeostasis

Resistance to traditional antifungal drugs has increased significantly over the past three decades, making identification of novel antifungal agents and new targets an emerging priority. Based on the extraordinary zinc requirement of several fungal pathogens and their well-established sensitivity to zinc deprivation, we developed an efficient cell-based screen to identify new antifungal drugs that target the zinc homeostasis machinery. The screen is based on the zinc-regulated transcription factor Zap1 of Saccharomyces cerevisiae, which regulates transcription of genes like the high-affinity zinc transporter ZRT1. We generated a genetically modified strain of S. cerevisae that reports intracellular zinc deficiency by placing the coding sequence of green fluorescent protein (GFP) under the control of the Zap1-regulated ZRT1 promoter. After showing that the GFP fluorescence signal correlates with low intracellular zinc concentrations in this strain, a protocol was developed for screening small-molecule libraries for compounds that induce Zap1-dependent GFP expression. Comparison of control compounds and known modulators of metal metabolism from the library reveals a robust screen (Z′ = 0.74) and validates this approach to the discovery of new classes of antifungal compounds that interfere with the intracellular zinc homeostasis. Given that growth of many pathogenic organisms is significantly impaired by zinc limitation; these results identify new types of antifungal drugs that target critical nutrient acquisition pathways

Heterologous Overexpression and Mutagenesis of the Human Bile Salt Export Pump (ABCB11) Using DREAM (Directed REcombination-Assisted Mutagenesis)

Homologous recombination in Saccharomyces cerevisiae is a well-studied process. Here, we describe a yeast-recombination-based approach to construct and mutate plasmids containing the cDNA of the human bile salt export pump (BSEP) that has been shown to be unstable in E. coli. Using this approach, we constructed the necessary plasmids for a heterologous overexpression of BSEP in the yeast Pichia pastoris. We then applied a new site-directed mutagenesis method, DREAM (Directed REcombination-Assisted Mutagenesis) that completely bypasses E. coli by using S. cerevisiae as the plasmid host with high mutagenesis efficiency. Finally, we show how to apply this strategy to unstable non-yeast plasmids by rapidly turning an existing mammalian BSEP expression construct into a S. cerevisiae-compatible plasmid and analyzing the impact of a BSEP mutation in several mammalian cell lines