Large-budded Cells Research Articles

Epistasis, aneuploidy, and functional mutations underlie evolution of resistance to induced microtubule depolymerization.

Cells with blocked microtubule polymerization are delayed in mitosis, but eventually manage to proliferate despite substantial chromosome missegregation. While several studies have analyzed the first cell division after microtubule depolymerization, we have asked how cells cope long-term with microtubule impairment. We allowed 24 clonal populations of yeast cells with beta-tubulin mutations preventing proper microtubule polymerization, to evolve for ˜150 generations. At the end of the laboratory evolution experiment, cells had regained the ability to form microtubules and were less sensitive to microtubule-depolymerizing drugs. Whole-genome sequencing identified recurrently mutated genes, in particular for tubulins and kinesins, as well as pervasive duplication of chromosome VIII. Recreating these mutations and chromosome VIII disomy prior to evolution confirmed that they allow cells to compensate for the original mutation in beta-tubulin. Most of the identified mutations did not abolish function, but rather restored microtubule functionality. Analysis of the temporal order of resistance development in independent populations repeatedly revealed the same series of events: disomy of chromosome VIII followed by a single additional adaptive mutation in either tubulins or kinesins. Since tubulins are highly conserved among eukaryotes, our results have implications for understanding resistance to microtubule-targeting drugs widely used in cancer therapy.

Defects in base excision repair and homologous recombination, but not nucleotide excision repair, lead to altered cell cycle phase distributions in Saccharomyces cerevisiae

The DNA within cells is constantly subjected to chemical modifications resulting from exposure to endogenous and exogenous DNA damaging agents. Damaged or oxidized bases are primarily repaired by the base excision repair (BER) pathway, crosslinks induced by ultraviolet (UV) light and many bulky lesions are repaired by nucleotide excision repair (NER), mismatches are resolved by the mismatch repair system (MMR), and double‐strand breaks in DNA are repaired by either the homologous recombination (HR) pathway or the nonhomologous end‐joining (NHEJ) pathway. Inactivation of NER genes such as RAD2, RAD7 or RAD14 leads to extreme UV sensitivity in yeast cells. Mutants with defects in BER genes OGG1 or UNG1 are defective in repair of oxidized guanines and the removal of uracil bases from DNA, respectively. Yeast rad52 mutants are deficient in HR and are hypersensitive to ionizing radiation and chemicals that induce double‐strand breaks.Our laboratory has previously observed that rad52 haploid and diploid mutant cell cultures have high levels of distended large‐budded G2 phase cells and long cell cycle transit times (see poster by C. England et al.). This work also demonstrated that the high G2 phase cell phenotype of rad52 cells was abolished when any of seven known DNA damage checkpoint genes were co‐inactivated. The current project has extended these studies and asked whether defects in other DNA repair pathways also lead to accumulation of G2 phase cells and increases in doubling times. Initial studies revealed that log phase cultures of mutants deficient in NER (rad2, rad7 and rad14) or MMR (msh2, msh3 and msh6) do not have high levels of large‐budded cells. By contrast, such cells were strongly elevated in mutant cultures defective in the BER pathway (ogg1 and ung1 cells). Recent experiments are attempting to determine the fraction of these large‐budded cells that are G2 vs. M phase cells via DAPI staining and whether they are specifically associated with activation of the DNA damage checkpoint system. Cell cultures containing mutants defective in other DNA repair processes, such as Rad6‐mediated postreplication repair, translesion synthesis and nuclease processing of DNA ends, are also being analyzed using phase contrast and fluorescence microscopy to identify unusual cell morphology and cell cycle phase characteristics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The PCNA binding domain of Rad2p plays a role in mutagenesis by modulating the cell cycle in response to DNA damage

The xeroderma pigmentosum group G (XPG) gene, encoding an essential element in nucleotide excision repair (NER), has a proliferating cell nuclear antigen-binding domain (PCNA-BD) at its C-terminal region. However, the role of this domain is controversial because its presence does not affect NER. Using yeast RAD2, a homolog of human XPG, we show that Rad2p interacts with PCNA through its PCNA-BD and the PCNA-BD of Rad2p plays a role in UV-induced mutagenesis. While a mutation of Rad2p endonuclease activity alone causes dramatically increased mutation rates and UV sensitivity, as well as growth retardation after UV irradiation, a mutation of the Rad2p PCNA-BD in the same mutant causes dramatically decreased mutation rates, reduced UV sensitivity and increased growth rate after UV irradiation. After UV irradiation, large-budded cells of Rad2p endonuclease defective mutants wane due to a mutation of the Rad2p PCNA-BD. Besides, the Rad2p PCNA-BD mutant protein exhibits alleviated PCNA-binding efficiency. These results show a hitherto unsuspected role of the Rad2p PCNA-BD that controls mutagenesis via cell cycle modulation together with PCNA. Furthermore, the high mutation rate of cells with other NER gene mutations was also decreased by the mutation of the Rad2p PCNA-BD, which indicates that the Rad2p-PCNA interaction might be responsible for mutagenesis control in the general NER pathway. Our results suggest that the drastically increased incidence of skin cancer in xeroderma pigmentosum patients could arise from the synergistic effects between cell cycle arrest due to the XPG-PCNA interaction and the accumulation of damaged DNA via defects in DNA damage repair.

Drug-Sensitive DNA Polymerase δ Reveals a Role for Mismatch Repair in Checkpoint Activation in Yeast

We have used a novel method to activate the DNA damage S-phase checkpoint response in Saccharomyces cerevisiae to slow lagging-strand DNA replication by exposing cells expressing a drug-sensitive DNA polymerase δ (L612M-DNA pol δ) to the inhibitory drug phosphonoacetic acid (PAA). PAA-treated pol3-L612M cells arrest as large-budded cells with a single nucleus in the bud neck. This arrest requires all of the components of the S-phase DNA damage checkpoint: Mec1, Rad9, the DNA damage clamp Ddc1-Rad17-Mec3, and the Rad24-dependent clamp loader, but does not depend on Mrc1, which acts as the signaling adapter for the replication checkpoint. In addition to the above components, a fully functional mismatch repair system, including Exo1, is required to activate the S-phase damage checkpoint and for cells to survive drug exposure. We propose that mismatch repair activity produces persisting single-stranded DNA gaps in PAA-treated pol3-L612M cells that are required to increase DNA damage above the threshold needed for checkpoint activation. Our studies have important implications for understanding how cells avoid inappropriate checkpoint activation because of normal discontinuities in lagging-strand replication and identify a role for mismatch repair in checkpoint activation that is needed to maintain genome integrity.

Transport to the plasma membrane is regulated differently early and late in the cell cycle in Saccharomyces cerevisiae

Traffic from the trans-Golgi network to the plasma membrane is thought to occur through at least two different independent pathways. The chitin synthase Chs3p requires the exomer complex and Arf1p to reach the bud neck of yeast cells in a cell-cycle-dependent manner, whereas the hexose transporter Hxt2p localizes over the entire plasma membrane independently of the exomer complex. Here, we conducted a visual screen for communalities and differences between the exomer-dependent and exomer-independent transport to the plasma membrane in Saccharomyces cerevisiae. We found that most of the components that are required for the fusion of transport vesicles with the plasma membrane, are involved in localization of both Chs3p and Hxt2p. However, the lethal giant larva homologue Sro7p is required primarily for the targeting of Chs3p, and not Hxt2p or other cargoes such as Itr1p, Cwp2p and Pma1p. Interestingly, this transport defect was more pronounced in large-budded cells just before cytokinesis than in small-budded cells. In addition, we found that the yeast Rab11 homologue Ypt31p determines the residence time of Chs3p in the bud neck of small-budded, but not large-budded, cells. We propose that transport to and from the bud neck is regulated differently in small- and large-budded cells, and differs early and late in the cell cycle.

A Role for the Karyopherin Kap123p in Microtubule Stability

Several components of the nuclear transport machinery play a role in mitotic spindle assembly in higher eukaryotes. To further investigate the role of this family of proteins in microtubule function, we screened for mutations in Saccharomyces cerevisiae that confer sensitivity to microtubule-destabilizing drugs. One mutant exhibiting this phenotype lacked the gene encoding the karyopherin Kap123p. Analysis of kap123Delta cells revealed that the drug sensitivity was caused by a defect in microtubule stability and/or assembly. In support of this idea, we demonstrated genetic interactions between the kap123Delta mutation and mutated alleles of genes encoding alpha-tubulins and factors controlling microtubule dynamics. Moreover, kap123Delta cells exhibit defects in spindle structure and dynamics as well as nuclear positioning defects during mitosis. Cultures of kap123Delta strains are enriched for mononucleated large-budded cells often containing short spindles and nuclei positioned away from the budneck, phenotypes indicative of defects in both cytoplasmic and nuclear microtubules. Finally, we identified a gene, CAJ1, which when deleted in combination with KAP123 exacerbated the microtubule-related defects of the kap123Delta mutants. We propose that Kap123p and Caj1p, a member of the Hsp40 family of proteins, together play an essential role in normal microtubule function.

WRN helicase defective in the premature aging disorder Werner syndrome genetically interacts with topoisomerase 3 and restores the top3 slow growth phenotype of sgs1 top3

Werner syndrome (WS) is a premature aging disorder characterized by genomic instability. The WRN gene defective in WS encodes a protein with both helicase and exonuclease activities that interacts with proteins implicated in DNA metabolism. To understand its genetic functions, we examined the ability of human WRN to rescue phenotypes associated with sgs1, the sole RecQ helicase in Saccharomyces cerevisiae. WRN failed to rescue sgs1 sensitivity to the DNA damaging agent methylmethane sulfonate or replication inhibitor hydroxyurea, suggesting divergent functions of human and yeast RecQ helicases. However, physiological expression of WRN in sgs1 top3 restored top3 slow growth phenotype, whereas no effect on growth was observed with wild-type or sgs1 strains. Slow growth of WRN-transformed sgs1 top3 correlated with an elevated population of large-budded cells with undivided nuclei, indicating restoration of cell cycle delay in late S/G2 characteristic of top3. WRN helicase but not exonuclease activity was genetically required for restoration of top3 growth phenotype, demonstrating separation of function of WRN catalytic activities. A naturally occurring missense polymorphism in WRN that interferes with helicase activity abolished its ability to restore top3 slow growth phenotype. Proposed roles of WRN in genetic pathways important for the suppression of genomic instability are discussed.

Checkpoint proteins control morphogenetic events during DNA replication stress in Saccharomyces cerevisiae

In response to DNA replication stress in Saccharomyces cerevisiae, the DNA replication checkpoint maintains replication fork stability, prevents precocious chromosome segregation, and causes cells to arrest as large-budded cells. The checkpoint kinases Mec1 and Rad53 act in this checkpoint. Treatment of mec1 or rad53Δ mutants with replication inhibitors results in replication fork collapse and inappropriate partitioning of partially replicated chromosomes, leading to cell death. We describe a previously unappreciated function of various replication stress checkpoint proteins, including Rad53, in the control of cell morphology. Checkpoint mutants have aberrant cell morphology and cell walls, and show defective bud site selection. Rad53 shows genetic interactions with septin ring pathway components, and, along with other checkpoint proteins, controls the timely degradation of Swe1 during replication stress, thereby facilitating proper bud growth. Thus, checkpoint proteins play an important role in coordinating morphogenetic events with DNA replication during replication stress.

Block of the cell cycle of the yeast Saccharomyces cerevisiae by tyrphostin, an inhibitor of protein tyrosine kinase.

Tyrphostins are inhibitors of the epidermal growth factor receptor tyrosine kinase. To elucidate the biological function of protein tyrosine kinases in yeast cells, a mutant hypersensitive to tyrphostin was isolated and investigated for its response to the drug. The mutation was recessive and was designated tpt1 for tyrphostin hypersensitive. A tpt1 strain cannot grow in the presence of tyrphostin, implying that a biological process sensitive to tyrphostin is essential for cell growth. Microscopic observation indicated that large-budded cells were accumulated in the presence of the inhibitor. The results suggest the involvement of protein tyrosine phosphorylation in the cell cycle progression of Saccharomyces cerevisiae.

Swe1p Responds to Cytoskeletal Perturbation, Not Bud Size, in S. cerevisiae

S. cerevisiae cells must grow to a critical size in G1 in order to pass start and enter the cell cycle. A recent study proposed that in addition to the mother size control in G1, the bud must grow to a critical bud size in G2 in order to enter mitosis. Insufficient bud size would cause G2 arrest enforced by the mitotic inhibitor Swe1p, explaining previous findings that some perturbations that block bud growth also trigger Swe1p-dependent cell-cycle arrest. We tested the critical-bud-size hypothesis. We found that halting bud growth by inactivation of the myosin Myo2p did not trigger Swe1p-dependent arrest in budded cells, even when the buds were very small. Moreover, Swe1p did not affect cell-cycle progression in unstressed cells, even when bud size was decreased by overriding G1 size control. Actin depolymerization did cause Swe1p-dependent arrest in small-budded but not large-budded cells, as previously reported. However, we found that the key determinant of cell-cycle arrest in those circumstances was not bud size, but rather the relative abundance of the Swe1p mitotic inhibitor and the mitosis-promoting cyclins. Swe1p does not respond to insufficient bud size. Instead, actin stress empowers Swe1p to promote arrest. The effectiveness of Swe1p in promoting that arrest declines as cells progress through the cell cycle.

Paclitaxel‐induced microtubule stabilization causes mitotic block and apoptotic‐like cell death in a paclitaxel‐sensitive strain of Saccharomyces cerevisiae

Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.

Saccharomyces cerevisiae Bni4p directs the formation of the chitin ring and also participates in the correct assembly of the septum structure.

In Saccharomyces cerevisiae cytokinesis is efficiently achieved when a concerted series of events take place at the neck region, leading to septum formation. Here it is shown that Bni4p plays a crucial role in this process. Deltabni4 mutants contain normal amounts of chitin and show normal chitin synthase III (CSIII) activity, but are partially resistant to Calcofluor White (CFW), probably due to the striking pattern of chitin distribution. CFW vital staining shows that chitin is synthesized in daughter cells and that it is also asymmetrically deposited at the mother-side of the neck in large-budded cells. This specific pattern coincides with that of Chs4p and Chs3p proteins. Alternatively, staining of unbudded cultures confirmed that Bni4p directs early chitin ring assembly, but is no longer required for the chitin deposition that occurs late in the cell cycle at cytokinesis. Consequently, this work provides a strategy to genetically discriminate between the absence of chitin synthesis (Deltachs3 mutant) and failure in chitin ring assembly (Deltabni4 mutants). The characterization of double mutants affected in chitin synthesis and primary septum (PS) assembly (Deltamyo1 and Deltachs2) provides evidence for the cooperation of Bni4p in PS formation besides its role in chitin ring assembly. In addition, it is shown that the chitin ring, but not the late deposition of chitin, cooperates in the correct assembly of the actomyosin ring and the PS when the biological function of the septins is compromised. We conclude that Bni4p is not only required for the assembly of the chitin ring, but is also involved in septum architecture and the maintenance of neck integrity.

Local exposure of phosphatidylethanolamine on the yeast plasma membrane is implicated in cell polarity.

Cell surface phosphatidylethanolamine (PE) of the yeast cell was probed by biotinylated Ro09-0198 (Bio-Ro), which specifically binds to PE and was visualized with fluorescein-labelled streptavidin. In Saccharomyces cerevisiae, the signals were observed at the presumptive bud site, the emerging small bud cortex, the bud neck of the late mitotic large-budded cells and the tip of the mating projection. In Schizosaccharomyces pombe, the signals were observed at one end or both ends of mono-nucleated cells and the division plane of the late mitotic cells. These sites were polarized ends in the yeast cells, implying that PE is exposed on the cell surface at cellular polarized ends. Treatment of S. cerevisiae cells with Ro09-0198 resulted in aberrant F-actin accumulation at the above sites, implying that limited surface exposure of PE is involved in the polarized organization of the actin cytoskeleton. Furthermore, S. cerevisiae ros3, dnf1 and dnf2 null mutants, which were known to be defective in the internalization of fluorescence-labelled PE, as well as the combinatorial mutants, were stained with Bio-Ro at the enlarging bud cortex, in addition to the Bio-Ro-staining sites of wild-type cells, suggesting that Ros3p, Dnf1p and Dnf2p are involved in the retrieval of exposed PE at the bud cortex.

The Deubiquitinating Enzyme Doa4p Protects Cells from DNA Topoisomerase I Poisons

DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.

Role for lipid signaling and the cell integrity MAP kinase cascade in yeast septum biogenesis.

Polarized deposition of chitin at the bud neck is essential for cell separation in yeast. Chitin septum biogenesis is catalyzed by two distinct chitin synthase activities encoded by the CHS2 and CHS3 genes. The phosphoinositide phosphatase Sac1p is required for proper trafficking of the Chs3p chitin synthase. sac1 mutants also display a severe synthetic growth defect, with mutations in the SLT2 gene which encodes a MAP kinase involved in cell integrity. We characterized the defect that underlies this genetic interaction and found that sac1 Delta slt2 Delta cells arrest as large-budded cells because they fail to separate at the end of mitosis. This inability to complete cell division appears to be caused by an increased deposition of chitin at the septum area and correlates with a mislocalized accumulation of the Chs2p chitin synthase at the cell periphery. Our data therefore indicate that Sac1p and Slt2p have synergistic roles in regulating chitin septum biogenesis.

Conservation of Mechanisms Controlling Entry into Mitosis: Budding Yeast Wee1 Delays Entry into Mitosis and Is Required for Cell Size Control

Background: In fission yeast, the Wee1 kinase delays entry into mitosis until a critical cell size has been reached; however, a similar role for Wee1-related kinases has not been reported in other organisms. SWE1, the budding yeast homolog of wee1, is thought to function in a morphogenesis checkpoint that delays entry into mitosis in response to defects in bud morphogenesis.Results: In contrast to previous studies, we found that budding yeast swe1Δ cells undergo premature entry into mitosis, leading to birth of abnormally small cells. Additional experiments suggest that conditions that activate the morphogenesis checkpoint may actually be activating a G2/M cell size checkpoint. For example, actin depolymerization is thought to activate the morphogenesis checkpoint by inhibiting bud morphogenesis. However, actin depolymerization also inhibits bud growth, suggesting that it could activate a cell size checkpoint. Consistent with this possibility, we found that actin depolymerization fails to induce a G2/M delay once daughter buds pass a critical size. Other conditions that activate the morphogenesis checkpoint block bud formation, which could also activate a size checkpoint if cell size at G2/M is monitored in the daughter bud. Previous work reported that Swe1 is degraded during G2, which was proposed to account for failure of large-budded cells to arrest in response to actin depolymerization. However, we found that Swe1 is present throughout G2 and undergoes hyperphosphorylation as cells enter mitosis, as found in other organisms.Conclusions: Our results suggest that the mechanisms known to coordinate entry into mitosis in other organisms have been conserved in budding yeast.

Hyphal tip-associated localization of Cdc42 is F-actin dependent in Candida albicans.

The rho-type GTPase Cdc42 is important for the establishment and maintenance of eukaryotic cell polarity. To examine whether Cdc42 is regulated during the yeast-to-hypha transition in Candida albicans, we constructed a green fluorescence protein (GFP)-Cdc42 fusion under the ACT1 promoter and observed its localization in live C. albicans cells. As in Saccharomyces cerevisiae, GFP-Cdc42 was observed around the entire periphery of the cell. In yeast-form cells of C. albicans, it clustered to the tips and sides of small buds as well as to the mother-daughter neck region of large-budded cells. Upon hyphal induction, GFP-Cdc42 clustered to the site of hyphal evagination and remained at the tips of the hyphae. This temporal and spatial localization of Cdc42 suggests that its activity is regulated during the yeast-to-hypha transition. In addition to the accumulation at the hyphal tip, GFP-Cdc42 was also seen as a band within the hyphal tube in cells that had undergone nuclear separation. With the F-actin-assembly inhibitor latrunculin A, we found that GFP-Cdc42 accumulation at the bud site in yeast-form cells is F-actin independent, whereas GFP-Cdc42 accumulation at the hyphal tip requires F-actin. Furthermore, disruption of the F-actin cytoskeleton impaired the transcriptional induction of hypha-specific genes. Therefore, hypha formation resembles mating in Saccharomyces cerevisiae in that both require F-actin for GFP-Cdc42 localization and efficient signaling.

The CENP-A homolog CaCse4p in the pathogenic yeast Candida albicans is a centromere protein essential for chromosome transmission.

The gene encoding CaCse4p, a homolog of the evolutionarily conserved histone H3-like kinetochore protein CENP-A, has been cloned from the human pathogenic diploid yeast Candida albicans. To study the phenotype of C. albicans diploid cells depleted of CaCse4p, we deleted one copy of CaCSE4 and brought the other copy under control of a regulated PCK1 promoter (repressed by glucose and induced by succinate). Inability of this strain to grow on glucose medium indicates that CaCse4p is essential for cell viability. Shutdown of CaCSE4 expression resulted in a sharp decline of CaCse4p levels with concomitant loss of cell viability. Examination of these CaCse4p-depleted cells revealed a mitosis-specific arrest phenotype with accumulation of large-budded cells containing single G(2) nuclei at or near the bud neck along with short mitotic spindles. Subcellular localization of CaCse4p by anti-CaCse4p antibodies in both budding and filamentous C. albicans cells revealed an intense dot-like signal always colocalized with 4',6-diamidino-2-phenylindole-stained nuclei. Unlike higher eukaryotes but similar to the budding yeast Saccharomyces cerevisiae, centromere separation in the budding yeast form of C. albicans occurs before anaphase, at a very early stage of the cell cycle. In the filamentous mode of cell division, however, centromere separation appears to occur in early anaphase. Coimmunostaining with anti-CaCse4p and antitubulin antibodies shows that CaCse4p localizes near spindle pole bodies, analogous to the localization pattern observed for kinetochore proteins in S. cerevisiae. CaCse4p promises to be a highly useful reagent for the study of centromere/kinetochore structure in C. albicans.

Role of SGS1 and SLX4 in maintaining rDNA structure in Saccharomyces cerevisiae.

To address the role of SGS1 in controlling genome stability we previously identified several slx mutants that require SGS1 for viability. Here, we report the isolation and characterization of temperature-sensitive (ts) SGS1 alleles in cells lacking SLX4. At the non-permissive temperature (37 degrees C) sgs1-ts slx4 cells progress through S-phase and arrest growth as large-budded cells with at least a 2C DNA content. Analysis of the integrity of the replicated DNA by pulsed-field gel electrophoresis revealed that chromosome XII (ChrXII) was uniquely altered, as it was unable to enter the gel. This defect was specific to the tandem rDNA repeats on ChrXII and occurred as cells progressed through S-phase at 37 degrees C. Reciprocal-shift experiments revealed that viability and ChrXII migration can be restored by allowing Sgs1 to act between G2/M and the subsequent G1 phase. These results suggest that Sgs1 and Slx4 are not required for bulk DNA synthesis but play redundant roles in maintaining rDNA structure during DNA replication.

An α-Tubulin Mutant Demonstrates Distinguishable Functions Among the Spindle Assembly Checkpoint Genes inSaccharomyces cerevisiae

AbstractCells expressing a mutant allele of α-tubulin, tub1-729, are cold sensitive and arrest as large-budded cells with microtubule defects. The cold sensitivity of tub1-729 is suppressed by extra copies of a subset of the mitotic checkpoint genes BUB1, BUB3, and MPS1, but not MAD1, MAD2, and MAD3. This suppression by checkpoint genes does not depend upon their role in the MAD2-dependent spindle assembly checkpoint. In addition, BUB1 requires an intact kinase domain as well as Bub3p to suppress tub1-729. The data suggest that tub1-729 cells are defective in microtubule-kinetochore attachments and that the products of specific checkpoint genes can act either directly or indirectly to affect these attachments.