Alison E. Gammie

Functional characterization of pathogenic human MSH2 missense mutations in Saccharomyces cerevisiae.

Hereditary nonpolyposis colorectal cancer (HNPCC) is associated with defects in DNA mismatch repair. Mutations in either hMSH2 or hMLH1 underlie the majority of HNPCC cases. Approximately 25% of annotated hMSH2 disease alleles are missense mutations, resulting in a single change out of 934 amino acids. We engineered 54 missense mutations in the cognate positions in yeast MSH2 and tested for function. Of the human alleles, 55% conferred strong defects, 8% displayed intermediate defects, and 38% showed no defects in mismatch repair assays. Fifty percent of the defective alleles resulted in decreased steady-state levels of the variant Msh2 protein, and 49% of the Msh2 variants lost crucial protein–protein interactions. Finally, nine positions are predicted to influence the mismatch recognition complex ATPase activity. In summary, the missense mutations leading to loss of mismatch repair defined important structure–function relationships and the molecular analysis revealed the nature of the deficiency for Msh2 variants expressed in the tumors. Of medical relevance are 15 human alleles annotated as pathogenic in public databases that conferred no obvious defects in mismatch repair assays. This analysis underscores the importance of functional characterization of missense alleles to ensure that they are the causative factor for disease.

Kar4p, a Karyogamy-Specific Component of the Yeast Pheromone Response Pathway

Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.

Mutation Rates, Spectra, and Genome-Wide Distribution of Spontaneous Mutations in Mismatch Repair Deficient Yeast

DNA mismatch repair is a highly conserved DNA repair pathway. In humans, germline mutations in hMSH2 or hMLH1, key components of mismatch repair, have been associated with Lynch syndrome, a leading cause of inherited cancer mortality. Current estimates of the mutation rate and the mutational spectra in mismatch repair defective cells are primarily limited to a small number of individual reporter loci. Here we use the yeast Saccharomyces cerevisiae to generate a genome-wide view of the rates, spectra, and distribution of mutation in the absence of mismatch repair. We performed mutation accumulation assays and next generation sequencing on 19 strains, including 16 msh2 missense variants implicated in Lynch cancer syndrome. The mutation rate for DNA mismatch repair null strains was approximately 1 mutation per genome per generation, 225-fold greater than the wild-type rate. The mutations were distributed randomly throughout the genome, independent of replication timing. The mutation spectra included insertions/deletions at homopolymeric runs (87.7%) and at larger microsatellites (5.9%), as well as transitions (4.5%) and transversions (1.9%). Additionally, repeat regions with proximal repeats are more likely to be mutated. A bias toward deletions at homopolymers and insertions at (AT)n microsatellites suggests a different mechanism for mismatch generation at these sites. Interestingly, 5% of the single base pair substitutions might represent double-slippage events that occurred at the junction of immediately adjacent repeats, resulting in a shift in the repeat boundary. These data suggest a closer scrutiny of tumor suppressors with homopolymeric runs with proximal repeats as the potential drivers of oncogenesis in mismatch repair defective cells.

Role of Transcription Factor Kar4 in Regulating Downstream Events in the Saccharomyces cerevisiae Pheromone Response Pathway

ABSTRACT Yeast Kar4 is a putative transcription factor required for karyogamy (the fusion of haploid nuclei during mating) and possibly other functions. Previously known to be required only for the transcriptional induction of KAR3 and CIK1, microarray experiments identified many genes regulated by Kar4 in both mating and mitosis. Several gene clusters are positively or negatively regulated by mating pheromone in a Kar4-dependent manner. Chromatin immunoprecipitation and gel shift assays confirmed that Kar4 binds to regulatory DNA sequences upstream of KAR3. Together with one-hybrid experiments, these data support a model in which both Kar4 and Ste12 bind jointly to the KAR3 promoter. Analysis of the upstream regions of Kar4-induced genes identified a DNA sequence motif that may be a binding site for Kar4. Mutation within the motif upstream of KAR3 eliminated pheromone induction. Genes regulated by Kar4, on average, are delayed in their temporal expression and exhibit a more stringent dose response to pheromone. Furthermore, the induction of Kar4 by pheromone is necessary for the delayed temporal induction of KAR3 and PRM2, genes required for efficient nuclear fusion during mating. Accordingly, we propose that Kar4 plays a critical role in the choreography of the mating response.

The Two Forms of Karyogamy Transcription Factor Kar4p Are Regulated by Differential Initiation of Transcription, Translation, and Protein Turnover

ABSTRACT Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatableGAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

Assays of cell and nuclear fusion

Publisher Summary This chapter discusses various assays of cell and nuclear fusion. The pathway of yeast mating forms a microcosm of the cell biological universe, encompassing fundamental problems in signaling, transcriptional regulation, polarization, motility, and membrane fusion. Each step in the pathway has been defined and dissected by the isolation and analysis of numerous mutations that cause specific defects in the efficiency of mating. Certain profoundly mating defective mutants may reduce the efficiency of mating by more than five orders of magnitude. Such mutants are generally called “sterile” or “ste” and are caused by defects in the initial pheromone signaling or the subsequent response pathways. In general, there are two basic ways to detect defects in cell and nuclear fusion. The first approach uses sensitive methods of detecting either the reduced efficiency of diploid formation or—in the case of nuclear fusion—the increased production of unique progeny cells called “cytoductants.” The second approach uses microscopic methods to detect the distinct zygotes in which cell or nuclear fusion has failed. It has the virtue of providing information about the nature of the mating defect but—because of the relatively fewer numbers of cells examined—may provide quantitatively less precise data.

Proteasome inhibition rescues clinically significant unstable variants of the mismatch repair protein Msh2

MSH2 is required for DNA mismatch repair recognition in eukaryotes. Deleterious mutations in human MSH2 account for approximately half of the alleles associated with a common hereditary cancer syndrome. Previously, we characterized clinically identified MSH2 missense mutations, using yeast as a model system, and found that the most common cause of defective DNA mismatch repair was low levels of the variant Msh2 proteins. Here, we show that increased protein turnover is responsible for the reduced cellular levels. Increasing gene dosage of more than half of the missense alleles fully restored function. A titration experiment revealed that raising the expression level of one variant to less than wild-type levels restored mismatch repair, suggesting that overexpression is not always required to regain function. We found that the ubiquitin-mediated proteasome degradation pathway is the major mechanism for increased turnover of the Msh2 variants and identified the primary ubiquitin ligase as San1. Deletion of San1 restored protein levels for all but one variant, but did not elevate wild-type Msh2 levels. The unstable variants interacted with San1, whereas wild-type Msh2 did not. Additionally, san1Δ suppressed the mismatch repair defect of unstable variants. Of medical significance, the clinically approved drug Bortezomib partially restored protein levels and mismatch repair function for low-level variants and reversed the resistance to cisplatin, a common chemotherapeutic. Our results provide the foundation for an innovative therapeutic regime for certain mismatch-repair-defective cancers that are refractory to conventional chemotherapies.

Reciprocal regulation of nuclear import of the yeast MutSα DNA mismatch repair proteins Msh2 and Msh6

DNA mismatch recognition is performed in eukaryotes by two heterodimers known as MutSalpha (Msh2/Msh6) and MutSbeta (Msh2/Msh3) that must reside in the nucleus to function. Two putative Msh2 nuclear localization sequences (NLS) were characterized by fusion to green fluorescent protein (GFP) and site-directed mutagenesis in the context of Msh2. One NLS functioned in GFP targeting assays and both acted redundantly within Msh2. We examined nuclear localization of each of the MutS monomers in the presence and absence of their partners. Msh2 translocated to the nucleus in cells lacking Msh3 and Msh6; however, cells lacking Msh6 showed significantly decreased levels of nuclear Msh2. Furthermore, the overall protein levels of Msh2 were significantly diminished in the absence of Msh6, particularly if Msh2 lacked a functional NLS. Msh3 localized in the absence of Msh2, but Msh6 localization depended on Msh2 expressing functional NLSs. Overall, the nuclear levels of Msh2 and Msh6 decline when the other partner is absent. The data suggest a stabilization mechanism to prevent free monomer accumulation in the cytoplasm.

Characterization of Pathogenic Human MSH2 Missense Mutations Using Yeast as a Model System: A Laboratory Course in Molecular Biology.

This work describes the project for an advanced undergraduate laboratory course in cell and molecular biology. One objective of the course is to teach students a variety of cellular and molecular techniques while conducting original research. A second objective is to provide instruction in science writing and data presentation by requiring comprehensive laboratory reports modeled on the primary literature. The project for the course focuses on a gene, MSH2, implicated in the most common form of inherited colorectal cancer. Msh2 is important for maintaining the fidelity of genetic material where it functions as an important component of the DNA mismatch repair machinery. The goal of the project has two parts. The first part is to create mapped missense mutation listed in the human databases in the cognate yeast MSH2 gene and to assay for defects in DNA mismatch repair. The second part of the course is directed towards understanding in what way are the variant proteins defective for mismatch repair. Protein levels are analyzed to determine if the missense alleles display decreased expression. Furthermore, the students establish whether the Msh2p variants are properly localized to the nucleus using indirect immunofluorescence and whether the altered proteins have lost their ability to interact with other subunits of the MMR complex by creating recombinant DNA molecules and employing the yeast 2-hybrid assay.

Characterization of the recA gene of Vibrio anguillarum

We have cloned and sequenced the recA gene from two strains, 775 and 531A, of the fish pathogen, Vibrio anguillarum. Although both strains showed different sensitivities to methyl methanesulfonate (MMS), the recA genes were identical. In vitro expression of the V. anguillarum recA gene produced a polypeptide of about 40 kDa, in agreement with the value obtained from the nucleotide sequence. We identified the transcription start point by primer extension. The promoter for the recA gene mapped to an SOS regulatory element. The presence of an SOS box suggests that a LexA-like mediated response system may exist in V. anguillarum. The deduced RecA amino acid sequence is highly homologous with Escherichia coli RecA and other RecA proteins. Domains important in RecA function are conserved. We provide a comparative analysis of the activities and features of RecA analogs from a variety of species. We observed that certain residues that could be important in protein conformation are conserved in RecA proteins across a diverse range of bacterial species.