Involvement of DNA End-Binding Protein Ku in Ty Element Retrotransposition

Saccharomyces cerevisiae Ty elements are retrotransposons whose life cycles are strikingly similar to those of retroviruses. They transpose via an RNA intermediate that is converted to linear double-stranded cDNA and then inserted into the host genome. Although Ty integration is mediated by the element-encoded integrase, it has been proposed that host factors are involved in this process. Here, we show that the DNA end-binding protein Ku, which functions in DNA double-strand break repair, potentiates retrotransposition. Specifically, by using a galactose-inducible Ty1 system, we found that in vivo, Ty1 retrotransposition rates were substantially reduced in the absence of Ku. In contrast, this phenotype was not observed with yeast strains containing mutations in other genes that are involved in DNA repair. We present evidence that Ku associates with Ty1 viruslike particles both in vitro and in vivo. These results provide an additional role for Ku and suggest that it might function in the life cycles of retroelements in other systems.     

Budding Yeast for Budding Geneticists: A Primer on the Saccharomyces cerevisiae Model System

The budding yeast Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of eukaryotic cell biology. This Primer article presents a brief historical perspective on the emergence of this organism as a premier experimental system over the course of the past century. An overview of the central features of the S. cerevisiae genome, including the nature of its genetic elements and general organization, is also provided. Some of the most common experimental tools and resources available to yeast geneticists are presented in a way designed to engage and challenge undergraduate and graduate students eager to learn more about the experimental amenability of budding yeast. Finally, a discussion of several major discoveries derived from yeast studies highlights the far-reaching impact that the yeast system has had and will continue to have on our understanding of a variety of cellular processes relevant to all eukaryotes, including humans.     

Cdc5 Interacts with the Wee1 Kinase in Budding Yeast

Development of a multicellular organism requires that mitosis and morphogenesis be coordinated. These processes must also be synchronized during the growth of unicellular organisms. In the yeast Saccharomyces cerevisiae, mitosis is dependent on the prior growth of a daughter cell in the form of a bud. Overexpression of wild-type Polo-like kinase Cdc5 or a catalytically inactive form resulted in the formation of multinucleate cells in budding yeast. Immunofluorescence analysis of these multinulceate cells showed that mitosis and bud formation were no longer linked. Others have shown that Swe1 is required for coupling mitosis to bud formation during a perturbed cell cycle. When the normal pathway of bud formation is perturbed, Swe1 functions to delay mitosis through negative regulation of Clb/Cdk. In cells lacking Swe1, multinucleate cells are formed in response to delays in bud formation. Affinity purification, two-hybrid analysis, and mutant characterization results suggested that Cdc5 and Swe1 interact. From these results, we conclude that multinucleate formation in response to Cdc5 overexpression is linked to titration of Swe1 function. These results also suggest that Cdc5 may be a negative regulator of Swe1.     

Centromere-Like Regions in the Budding Yeast Genome

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres.     

The Role of Interelement Selection in Saccharomyces cerevisiae Ty Element Evolution

Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes. The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside. Elements with higher rates of genomic replication possess a selective advantage over less active elements. Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements. To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed. The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data. In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons. The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies. These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages. We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside. Received: 5 November 1998 / Accepted: 17 March 1999     

The Role of Replication Bypass Pathways in Dicentric Chromosome Formation in Budding Yeast

Gross chromosomal rearrangements (GCRs) are large scale changes to chromosome structure and can lead to human disease. We previously showed in Saccharomyces cerevisiae that nearby inverted repeat sequences (∼20–200 bp of homology, separated by ∼1–5 kb) frequently fuse to form unstable dicentric and acentric chromosomes. Here we analyzed inverted repeat fusion in mutants of three sets of genes. First, we show that genes in the error-free postreplication repair (PRR) pathway prevent fusion of inverted repeats, while genes in the translesion branch have no detectable role. Second, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Third, we provide evidence for a faulty template switch model by studying mutants of DNA polymerases; defects in DNA pol delta (lagging strand polymerase) and Mgs1 (a pol delta interacting protein) lead to a defect in fusion events as well as allelic recombination. Pol delta and Mgs1 may collaborate either in strand annealing and/or DNA replication involved in fusion and allelic recombination events. Fourth, by studying genes implicated in suppression of GCRs in other studies, we found that inverted repeat fusion has a profile of genetic regulation distinct from these other major forms of GCR formation.ALL organisms are prone to large-scale changes (gross chromosomal rearrangements, GCRs) to their genomes that include deletions, inversions, and translocations. These large-scale changes are thought to drive evolutionary events, such as speciation, and contribute to human pathology such as Pelziaeus-Merzbacher syndrome and other genetic disorders (Lee et al. 2007; Stankiewicz and Lupski 2010). Thus, a firm understanding of how cells normally prevent such rearrangements, and how they accumulate, is critical to our understanding of both evolution and pathology.GCRs arise by many different mechanisms, and there is growing evidence that errors during DNA replication are a major source (Myung et al. 2001; Admire et al. 2006; Mizuno et al. 2009). Errors are thought to arise when replication forks encounter “lesions” on the template strand. Lesions can consist of protein complexes bound to DNA or lesions in the DNA itself. Replication forks bypass lesions by several different mechanisms that are still poorly understood (Atkinson and McGlynn 2009; Weinert et al. 2009). We believe that understanding lesion bypass mechanisms is central to understanding both how GCRs are prevented and how they form when lesion bypass mechanisms fail.All lesion bypass pathways utilize sequence homology to restart replication (Atkinson and McGlynn 2009; Weinert et al. 2009). Use of sequence homology during restart may limit the frequency of GCRs, as it lowers the probability of annealing to nonallelic sequences. Repetitive sequences present a problem because lesion bypass at sites near repetitive sequences may lead to annealing of nonallelic sequences and thus to GCR formation (Lemoine et al. 2005; Narayanan et al. 2006; Argueso et al. 2008). Indeed in yeast and in other organisms, GCRs occur frequently in repeat sequences (Dunham et al. 2002; Argueso et al. 2008; Di Rienzi et al. 2009). Some rearrangements do occur between so-called “single-copy sequences” with either no homology or limited homology (microhomologies of 5–9 bp; Myung et al. 2001; Kolodner et al. 2002; Putnam et al. 2005) though evidence suggests these rearrangements occur less frequently than rearrangements between repetitive sequences (Putnam et al. 2009). Interestingly, it has been shown that some genes are required to prevent the fusion of repetitive elements yet have no effect on rearrangements between single-copy sequences (Putnam et al. 2009). Currently it is not clear how these pathways act to suppress repeat-mediated events and why they are not required to prevent rearrangements between single-copy sequences.Our current understanding of the mechanisms underlying GCR formation is mostly derived from assays designed to detect specific changes to yeast chromosomes (Chen and Kolodner 1999; Myung et al. 2001; Huang and Koshland 2003; Lambert et al. 2005; Rattray et al. 2005; Admire et al. 2006; Narayanan et al. 2006; Schmidt et al. 2006; Smith et al. 2007; Pannunzio et al. 2008; Payen et al. 2008; Paek et al. 2009; Mizuno et al. 2009). Previously we reported on GCR formation in the budding yeast Saccharomyces cerevisiae using an assay we developed. We found that a major source of genome instability involves the fusion of nearby inverted repeats (with ∼20–200 bp of sequence homology, separated by 1–5 kb) to form either dicentric or acentric chromosomes (Figure 1D; Paek et al. 2009). We also found that fusion of inverted repeats is general: fusion occurred between inverted repeats at all five different locations tested on four different yeast chromosomes, as well as between synthetic inverted repeats (Paek et al. 2009). Genetic data suggest that these events most likely occur during replication of DNA (Admire et al. 2006). Further genetic analysis suggested that the mechanism of inverted repeat fusion differed from that of direct repeat recombination, in that inverted repeat fusion did not require genes involved in homologous recombination (HR) or single-strand annealing (SSA) pathways (Paek et al. 2009). In addition, fusion events are unlikely to involve double-strand breaks (DSBs), as genes in the nonhomologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) are not required for fusion events (Paek et al. 2009). Indeed gene knockouts in the HR (RAD52, RAD51, and RAD59), SSA (RAD52 and RAD1) and postreplication repair (PRR) (RAD18) pathways actually increased the frequency of fusion of an inverted repeat on chromosome (Chr) VII (Paek et al. 2009); these pathways normally suppress inverted repeat fusion.Open in a separate windowFigure 1.—Experimental setup for the detection of inverted repeat fusion and chromosome instability. Objects are not drawn to scale. (A) The starting strain has two copies of Chr VII. One copy contains the CAN1 gene, ADE6, ade3, while the other copy is ade6, ADE3. Cells are plated to canavanine, and three types of colonies are formed: (B) Allelic recombinants are round in appearance and are Ade⁺; (C) colonies that form by loss of Chr VII are round in appearance and Ade⁻; and (D) cells that contain unstable dicentric chromosomes form by the fusion of inverted repeats. One specific case of this fusion (the S2/S3 dicentric) is shown within braces. Cells with dicentrics form mixed colonies, which contain allelic recombinants, chromosome loss events, as well as a translocation between D7 and D11. The bar in the S2/S3 repeat represents a fusion junction. (E) The specific dicentric is detected by dicentric primers DP1 and DP2 and (F) a monocentric translocation that is detected with translocation primers TP1 and TP2.To further our previous studies, we analyzed three groups of genes implicated in the maintenance of genome stability. We tested how these genes affect the overall stability of Chr VII, focusing on the fusion of nearby inverted repeats to form a specific dicentric Chr VII and the resolution of the dicentric into a monocentric translocation (which we term the 403–535 translocation; Figure 1, D–F). First, we analyzed several genes in the PRR pathway and found that error-free bypass, but not translesion synthesis, is required for the prevention of inverted repeat fusion. Surprisingly, we found that siz1 mutants, which are defective for Srs2 recruitment to replication forks, and srs2 mutants had opposite effects on instability. This may reflect separate roles for Srs2 in different phases of the cell cycle. Second, we analyzed several mutations in genes that are associated with replication forks. We found that mutants in POL3 (polymerase delta) and MGS1 (encoding a single-strand annealing protein, which binds polymerase delta) significantly reduced the frequency of dicentric formation and allelic recombinants that arise in the checkpoint mutant rad9 (Giot et al. 1997; Hishida et al. 2001; Paek et al. 2009). Finally we studied genes associated with rearrangements involving repeats or single-copy sequences, as well as a subset of mutants involved in recombination. Generally, we find that the mechanisms of nearby inverted repeat fusion are distinct from mechanisms fusing longer repeats or single-copy sequences.     

Analysis of Yeast Retrotransposon Ty Insertions at the Can1 Locus

The target site distribution for 55 independent Ty insertions that inactivate the function of the Saccharomyces cerevisiae CAN1 gene is reported. Under some selection conditions Ty elements inserted preferentially into the promoter and exhibited an orientation bias. In contrast, under other conditions no insertions were detected in the promoter region and transposition appeared to occur randomly throughout the CAN1 coding sequence. These results show that the target site distribution for Ty insertions may be a function of the selection conditions.     

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice ¹. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage ². Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.Open in a separate windowClick here to view.^{(75M, flv)}     

Potassium Uptake Through the TOK1 K+ Channel in the Budding Yeast

The current through TOK1 (YKC1), the outward-rectifying K⁺ channel in Saccharomyces cerevisiae, was amplified by expressing TOK1 from a plasmid driven by a strong constitutive promoter. TOK1 so hyper-expressed could overcome the K⁺ auxotrophy of a mutant missing the two K⁺ transporters, TRK1 and TRK2. This trk1Δtrk2Δ double mutant hyperexpressing the TOK1 transgene had a higher internal K⁺ content than one expressing the empty plasmid. We examined protoplasts of these TOK1-hyperexpressing cells under a patch clamp. Besides the expected K⁺ outward current activating at membrane potential (V _m ) above the K⁺ equilibrium potential (E _K+ ), a small inward current was consistently observed when the V _m was slightly below E _K+ . The inward and the outward currents are similar in their activation rates, deactivation rates, ion specificities and Ba²⁺ inhibition, indicating that they flow through the same channel. Thus, the yeast outwardly rectifying K⁺ channel can take up K⁺ into yeast cells, at least under certain conditions. Received: 1 October 1998/Revised: 9 December 1998