The Autonomously Replicating Sequence (ARS) of the Yeast Saccharomyces exiguus Yp74L-3

Fragments containing ARSes were cloned from the genomic DNA of the yeast Saccharomyces exiguus Yp74L-3, and the essential regions for ARSes were restricted for these fragments. Mapping studies of ARS-acting sequences in one of these fragments suggested that S. exiguus recognizes a sequence as an ARS that is different from that recognized by Saccharomyces cerevisiae. Two ARS essential regions of S. exiguus were sequenced, and an ARS core consensus sequence of S. exiguus was deduced to be MATTAMWAWWTK. This sequence differs significantly from that of S. cerevisiae in two positions, suggesting that these nucleotide substitutions cause the difference in the ARS-recognition modes between S. exiguus and S. cerevisiae. Received: 3 August 1998 / Accepted: 18 September 1998     

Mechanisms Controlling the Integrity of Replicating Chromosomes in Budding Yeast

Cells are continually challenged by genomic insults that originate from chemical and physical agents diffused in the environment, but also normal cellular metabolism produces genotoxic effects. Moreover, DNA replication and recombination generate intermediates potentially dangerous for genome stability. Growing evidence show that many genetic disorders are characterized by high levels of chromosome alterations due to genomic instability, which is also a hallmark of cancer cells. Recent work shed some light on the molecular events that maintain the integrity of chromosomes during unperturbed S phase and in the face of odds.     

Helicase Activation and Establishment of Replication Forks at Chromosomal Origins of Replication

Many replication proteins assemble on the pre-RC-formed replication origins and constitute the pre-initiation complex (pre-IC). This complex formation facilitates the conversion of Mcm2–7 in the pre-RC to an active DNA helicase, the Cdc45–Mcm–GINS (CMG) complex. Two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK), work to complete the formation of the pre-IC. Each kinase is responsible for a distinct step of the process in yeast; Cdc45 associates with origins in a DDK-dependent manner, whereas the association of GINS with origins depends on CDK. These associations with origins also require specific initiation proteins: Sld3 for Cdc45; and Dpb11, Sld2, and Sld3 for GINS. Functional homologs of these proteins exist in metazoa, although pre-IC formation cannot be separated by requirement of DDK and CDK because of experimental limitations. Once the replicative helicase is activated, the origin DNA is unwound, and bidirectional replication forks are established.The main events at the initiation step of DNA replication are the unwinding of double-stranded DNA and subsequent recruitment of DNA polymerases, to start DNA synthesis. Eukaryotic cells require an active DNA helicase to unwind the origin DNA. The core components of the replicative helicase, Mcm2–7, are loaded as a head-to-head double hexamer connected via their amino-terminal rings (Evrin et al. 2009; Remus et al. 2009; Gambus et al. 2011) onto Orc-associated origins, to form the pre-RC in late M and G₁ phases (see Bell and Kaguni 2013). However, Mcm2–7 alone does not show DNA helicase activity at replication origins. After the formation of the pre-RC, other replication factors assemble on origins, and the pre-initiation complex (pre-IC) is formed. The pre-IC is defined as a complex formed just before the initiation of DNA replication (Zou and Stillman 1998); in yeast, it contains at least seven additional factors: Cdc45, GINS, Dpb11, Sld2, Sld3, Cdc45, and DNA polymerase ε (Pol ε) (Muramatsu et al. 2010). The formation of the pre-IC is a prerequisite for the activation of the Mcm2–7 helicase; two additional factors, Cdc45 and GINS, associate with Mcm2–7 and form a tight complex, the Cdc45–Mcm–GINS (CMG) complex (Gambus et al. 2006; Moyer et al. 2006). This reaction requires components of the pre-IC and two protein kinases, cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) (for reviews, see Labib 2010; Masai et al. 2010; Tanaka and Araki 2010). In this article, we summarize and discuss the manner via which the pre-IC is formed in yeasts and metazoa. Although there are some discrepancies, the process of formation of the pre-IC is conserved fairly well in these organisms.     

Self-Regulating Model for Control of Replication Origin Firing in Budding Yeast

A major research area concentrates on understanding the regulation of replication origin firing. It is now appreciated that checkpoint signaling participates in this controlled process and that defects in such signaling systems affect genome integrity. Inhibition of replication origin firing is most obviously apparent under conditions of replication stress, but origin firing must also be regulated on a minute-by-minute basis as cells progress normally through an unabated S-phase. Here we summarize a straightforward model to account for how origin firing could be controlled by a self-regulating system.     

Regulatory Mechanisms That Prevent Re-initiation of DNA Replication Can Be Locally Modulated at Origins by Nearby Sequence Elements

Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ∼60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others.     

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.     

Time to Be Versatile: Regulation of the Replication Timing Program in Budding Yeast

Eukaryotic replication origins are activated at different times during the S phase of the cell cycle, following a temporal program that is stably transmitted to daughter cells. Although the mechanisms that control initiation at the level of individual origins are now well understood, much less is known on how cells coordinate replication at hundreds of origins distributed on the chromosomes. In this review, we discuss recent advances shedding new light on how this complex process is regulated in the budding yeast Saccharomyces cerevisiae. The picture that emerges from these studies is that replication timing is regulated in cis by mechanisms modulating the chromatin structure and the subnuclear organization of origins. These mechanisms do not affect the licensing of replication origins but determine their ability to compete for limiting initiation factors, which are recycled from early to late origins throughout the length of the S phase.     

Reversion at the HIS1 Locus of Yeast

The his1 gene (chromosome V) of Saccharomyces cerevisiae specifies phosphoribosyl transferase (E.C.2.4.2.17), the first enzyme of histidine biosynthesis. This hexameric enzyme has both catalytic and regulatory functions. The spontaneous reversion rates of seven his1 mutations were studied. The reversion rates of the alleles at the proximal end of the locus (relative to the centromere) were about 50-fold higher than distal alleles. Spontaneous reversion to prototrophy was studied in diploids homoallelic for each of the seven his1 mutations. Based on tetrad analysis, the prototrophy revertants could be assigned to three classes: (1) revertant tetrads that carried a prototrophic allele indistinguishable from wild type; (2) revertant tetrads that carried a prototrophic allele characterized by histidine excretion and feedback resistance; and (3) revertant tetrads that did not contain a prototrophic spore, but rather a newly derived allele that complemented the original allele intragenically. Four of the seven his1 mutations produced the excretor revertant class, and two mutations produced the complementer revertant class. The significance of these findings to our understanding of gene organization and the catalytic and regulatory functions of gene products are discussed.     

Regulatory Mutants at the his1 Locus of Yeast

The his1 gene in Saccharomyces cerevisiae codes for phosphoribosyl transferase, an allosteric enzyme that catalyzes the initial step in histidine biosynthesis. Mutants that specifically alter the feedback regulatory function were isolated by selecting his1 prototrophic revertants that overproduce and excrete histidine. The prototrophs were obtained from diploids homoallelic for his1--7 and heterozygous for the flanking markers thr3 and arg6. Among six independently derived mutant isolates, three distinct levels of histidine excretion were detected. The mutants were shown to be second-site alterations mapping at the his1 locus by recovery of the original auxotrophic parental alleles. The double mutants, HIS1--7e, are dominant with respect to catalytic function but recessive in regulatory function. When removed from this his1--7 background, the mutant regulatory site (HIS1-e) still confers prototrophy but not histidine excretion. To yield the excretion phenotype, the primary and altered secondary sites are required in cis array. Differences in histidine excretion levels correlate with resistance to the histidine analogue, triazoalanine.     

Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast

A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism Saccharomyces cerevisiae. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.     

Novel Interallelic Complementation at the his1 Locus of Yeast

In yeast, 17 histidine-requiring mutants derived from and interallelically complementary to his1-7 were analyzed. The genetic basis of the complementation response was elucidated by mitotic and meiotic gene conversion. Each allele probably carries an unaltered 7-site mutation and a unique second-site alteration. The second-site alterations appear to be clustered within the proximal and distal segments of the his1 structural gene. Models of intraalelic complementation are reviewed in light of the unique complementational response between a single-site mutant and a double mutant including the identical altered base sequence.     

CDK-Dependent Nuclear Localization of B-Cyclin Clb1 Promotes FEAR Activation during Meiosis I in Budding Yeast

Cyclin-dependent kinases (CDK) are master regulators of the cell cycle in eukaryotes. CDK activity is regulated by the presence, post-translational modification and spatial localization of its regulatory subunit cyclin. In budding yeast, the B-cyclin Clb1 is phosphorylated and localizes to the nucleus during meiosis I. However the functional significance of Clb1''s phosphorylation and nuclear localization and their mutual dependency is unknown. In this paper, we demonstrate that meiosis-specific phosphorylation of Clb1 requires its import to the nucleus but not vice versa. While Clb1 phosphorylation is dependent on activity of both CDK and polo-like kinase Cdc5, its nuclear localization requires CDK but not Cdc5 activity. Furthermore we show that increased nuclear localization of Clb1 during meiosis enhances activation of FEAR (Cdc Fourteen Early Anaphase Release) pathway. We discuss the significance of our results in relation to regulation of exit from meiosis I.     

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.     

Symmetry Breaking in the Life Cycle of the Budding Yeast

The budding yeast Saccharomyces cerevisiae has been an invaluable model system for the study of the establishment of cellular asymmetry and growth polarity in response to specific physiological cues. A large body of experimental observations has shown that yeast cells are able to break symmetry and establish polarity through two coupled and partially redundant intrinsic mechanisms, even in the absence of any pre-existing external asymmetry. One of these mechanisms is dependent upon interplay between the actin cytoskeleton and the Rho family GTPase Cdc42, whereas the other relies on a Cdc42 GTPase signaling network. Integral to these mechanisms appear to be positive feedback loops capable of amplifying small and stochastic asymmetries. Spatial cues, such as bud scars and pheromone gradients, orient cell polarity by modulating the regulation of the Cdc42 GTPase cycle, thereby biasing the site of asymmetry amplification.The budding yeast Saccharomyces cerevisiae is a gift of nature, not just for its superb ability in fermentation to provide us food for hunger and pastime, but also for its relatively simple physiology, which has illuminated our understanding of many fundamental cellular processes. In particular, asymmetry is a way of life for the budding yeast, both when it grows vegetatively and initiates sexual reproductive cycles; as such, yeast has been an invaluable model for studying the establishment of cellular asymmetry. A haploid yeast cell in the G1 phase, which is round and grows isotropically, faces two options: to enter the mitotic cell cycle and grow a bud, or to refrain from cell cycle entry and form a mating projection (shmoo) toward a cell of the opposite mating type. In either case, the cell has to break symmetry to switch from isotropic growth to growth along a polarized axis (Fig. 1). These processes of cell polarity establishment are triggered either by internal signals from the cell cycle engine (budding) or by an external signal in the form of a pheromone gradient (mating).Open in a separate windowFigure 1.Symmetry breaking processes in the life cycle of budding yeast. Shown are the locations of actin patches, actin cables, and Cdc42 during polarized growth for both cycling cells and cells undergoing pheromone response. In G1 cells, Cdc42 is distributed symmetrically, and the actin cytoskeleton is not polarized. In response to cell cycle signals or mating pheromone stimulation, Cdc42 and the actin cytoskeleton become polarized: Cdc42 forms a “polar cap” and actin cables become oriented to allow for targeted secretion. Polarized growth further leads to formation of a bud (cell cycle signal) or formation of a mating projection (pheromone signal). Images represent GFP-Cdc42 (green), and rhodamine-phalloidin staining of filamentous actin (red).Pioneering work involving isolation and characterization of mutants deficient in various aspects of budding and shmoo formation identified key components of the molecular pathways underlying yeast polarized morphogenesis. Despite the relative simplicity of yeast, it has become increasingly clear that many of the genes that control the establishment of cell polarity are conserved between yeast and more complex eukaryotic organisms (see McCaffrey and Macara 2009; Munro and Bowerman 2009; Wang 2009; Nelson 2009). In particular, the small GTPase Cdc42, first discovered in yeast (Adams et al. 1990) and subsequently shown to be required for cell polarization in many eukaryotic organisms (Etienne-Manneville 2004), is the central regulator of yeast polarity.Common principles have begun to emerge to explain symmetry breaking under varying physiological conditions. One of these principles is the self-organizing nature of cell polarity. Whereas under physiological conditions yeast cells polarize toward an environmental asymmetry (pheromone gradient) or a “landmark,” i.e., the bud scar, deposited on the cell surface from a previous division (in a process called bud site selection), it is clear that the ability to undergo symmetry breaking to establish polarity in a random orientation is independent of these cues. It is tempting to speculate that the basic molecular machinery for symmetry breaking, which is required for asexual proliferation through budding, might have evolved independently of the machinery underlying mating and bud site selection.As in all polarized cell systems, yeast polarity is manifested as both an asymmetry in the distribution of signaling molecules and in the organization of the cytoskeleton. In yeast, the switch from an isotropic distribution of Cdc42 on the plasma membrane to a polarized distribution (Fig. 1) is required for the polarized organization of the actin cytoskeleton and membrane trafficking systems, and eventually orientated cell growth. Recent work also showed that the cytoskeleton and the membrane trafficking system can in turn impact the localization of Cdc42 and possibly other membrane‐associated regulatory molecules (Karpova et al. 2000; Wedlich-Soldner et al. 2004; Irazoqui et al. 2005; Zajac et al. 2005). A combination of experimental and theoretical analyses strongly suggests that the interplay between signaling and structural pathways is at the heart of the cell’s intrinsic ability to break symmetry.As there have been recent review articles on the polarized organization of budding yeast growth systems (Bretscher 2003; Pruyne et al. 2004b) and on the molecular parts list involved in cell polarization (Park and Bi 2007), this article is specifically focused on the mechanisms of symmetry breaking at two levels: first as a self-organization process accomplished through dynamic interplay between intrinsic signaling and cytoskeletal systems, which enables vegetative proliferation through bud formation; and second, as an adaptive process where polarity is spatially harnessed by physical cues that arise during bud-site selection and mating. Finally, we briefly extend our discussion to include the role of polarity in yeast aging and cell fate determination. This exciting, relatively new area of research has made important advances in our understanding of how asymmetry can be an important mechanism to ensure long-lasting fitness of a fast proliferating population.