Dad1p, Third Component of the Duo1p/Dam1p Complex Involved in Kinetochore Function and Mitotic Spindle Integrity

We showed recently that a complex between Duo1p and Dam1p is required for both spindle integrity and kinetochore function in the budding yeast Saccharomyces cerevisiae. To extend our understanding of the functions and interactions of the Duo1p/Dam1p complex, we analyzed the novel gene product Dad1p (for Duo1 and Dam1 interacting). Dad1p physically associates with Duo1p by two-hybrid analysis, coimmunoprecipitates with Duo1p and Dam1p out of yeast protein extracts, and shows interdependent localization with Duo1p and Dam1p to the mitotic spindle. These results indicate that Dad1p functions as a component of the Duo1p/Dam1p complex. Like Duo1p and Dam1p, Dad1p also localizes to kinetochore regions in chromosomes spreads. Here, we also demonstrate by chromatin immunoprecipitation that Duo1p, Dam1p, and Dad1p associate specifically with centromeric DNA in a manner that is dependent upon Ndc10 and partially dependent upon the presence of microtubules. To explore the functions of Dad1p in vivo, we generated a temperature-sensitive allele, dad1-1. This allele shows spindle defects and a mitotic arrest phenotype that is dependent upon the spindle assembly checkpoint. In addition, dad1-1 mutants undergo chromosome mis-segregation at the restrictive temperature, resulting in a dramatic decrease in viability.

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Implication of a novel multiprotein Dam1p complex in outer kinetochore function

The Journal of Cell Biology ◽

10.1083/jcb.200109063 ◽

2001 ◽

Vol 155 (7) ◽

pp. 1137-1146 ◽

Cited By ~ 133

Author(s):

Iain M. Cheeseman ◽

Christine Brew ◽

Michael Wolyniak ◽

Arshad Desai ◽

Scott Anderson ◽

...

Keyword(s):

Mitotic Spindle ◽

Fluorescent Protein ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants ◽

Phosphorylation Event ◽

Spindle Microtubules ◽

Green Fluorescent ◽

Kinetochore Function

Dam1p, Duo1p, and Dad1p can associate with each other physically and are required for both spindle integrity and kinetochore function in budding yeast. Here, we present our purification from yeast extracts of an ∼245 kD complex containing Dam1p, Duo1p, and Dad1p and Spc19p, Spc34p, and the previously uncharacterized proteins Dad2p and Ask1p. This Dam1p complex appears to be regulated through the phosphorylation of multiple subunits with at least one phosphorylation event changing during the cell cycle. We also find that purified Dam1p complex binds directly to microtubules in vitro with an affinity of ∼0.5 μM. To demonstrate that subunits of the Dam1p complex are functionally important for mitosis in vivo, we localized Spc19–green fluorescent protein (GFP), Spc34-GFP, Dad2-GFP, and Ask1-GFP to the mitotic spindle and to kinetochores and generated temperature-sensitive mutants of DAD2 and ASK1. These and other analyses implicate the four newly identified subunits and the Dam1p complex as a whole in outer kinetochore function where they are well positioned to facilitate the association of chromosomes with spindle microtubules.

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Mitotic Spindle Integrity and Kinetochore Function Linked by the Duo1p/Dam1p Complex

The Journal of Cell Biology ◽

10.1083/jcb.152.1.197 ◽

2001 ◽

Vol 152 (1) ◽

pp. 197-212 ◽

Cited By ~ 105

Author(s):

Iain M. Cheeseman ◽

Maria Enquist-Newman ◽

Thomas Müller-Reichert ◽

David G. Drubin ◽

Georjana Barnes

Keyword(s):

Mitotic Spindle ◽

Genetic Interactions ◽

Yeast Protein ◽

Yeast Saccharomyces Cerevisiae ◽

Chromosome Missegregation ◽

Shared Function ◽

Mutant Phenotypes ◽

Bipolar Spindles ◽

Kinetochore Function

Duo1p and Dam1p were previously identified as spindle proteins in the budding yeast, Saccharomyces cerevisiae. Here, analyses of a diverse collection of duo1 and dam1 alleles were used to develop a deeper understanding of the functions and interactions of Duo1p and Dam1p. Based on the similarity of mutant phenotypes, genetic interactions between duo1 and dam1 alleles, interdependent localization to the mitotic spindle, and Duo1p/Dam1p coimmunoprecipitation from yeast protein extracts, these analyses indicated that Duo1p and Dam1p perform a shared function in vivo as components of a protein complex. Duo1p and Dam1p are not required to assemble bipolar spindles, but they are required to maintain metaphase and anaphase spindle integrity. Immunofluorescence and electron microscopy of duo1 and dam1 mutant spindles revealed a diverse variety of spindle defects. Our results also indicate a second, previously unidentified, role for the Duo1p/Dam1p complex. duo1 and dam1 mutants show high rates of chromosome missegregation, premature anaphase events while arrested in metaphase, and genetic interactions with a subset of kinetochore components consistent with a role in kinetochore function. In addition, Duo1p and Dam1p localize to kinetochores in chromosome spreads, suggesting that this complex may serve as a link between the kinetochore and the mitotic spindle.

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A Novel Functional Domain of Cdc15 Kinase Is Required for Its Interaction With Tem1 GTPase in Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/157.4.1437 ◽

2001 ◽

Vol 157 (4) ◽

pp. 1437-1450 ◽

Cited By ~ 1

Author(s):

Kazuhide Asakawa ◽

Satoshi Yoshida ◽

Fumiaki Otake ◽

Akio Toh-e

Keyword(s):

Saccharomyces Cerevisiae ◽

Kinase Domain ◽

Binding Domain ◽

Temperature Sensitive ◽

Functional Domain ◽

Restrictive Temperature ◽

Cyclin Dependent Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Hybrid Screening

Abstract Exit from mitosis requires the inactivation of cyclin-dependent kinase (CDK) activity. In the budding yeast Saccharomyces cerevisiae, a number of gene products have been identified as components of the signal transduction network regulating inactivation of CDK (called the MEN, for the mitotic exit network). Cdc15, one of such components of the MEN, is an essential protein kinase. By the two-hybrid screening, we identified Cdc15 as a binding protein of Tem1 GTPase, another essential regulator of the MEN. Coprecipitation experiments revealed that Tem1 binds to Cdc15 in vivo. By deletion analysis, we found that the Tem1-binding domain resides near the conserved kinase domain of Cdc15. The cdc15-LF mutation, which was introduced into the Tem1-binding domain, reduced the interaction with Cdc15 and Tem1 and caused temperature-sensitive growth.The kinase activity of Cdc15 was not so much affected by the cdc15-LF mutation. However, Cdc15-LF failed to localize to the SPB at the restrictive temperature. Our data show that the interaction with Tem1 is important for the function of Cdc15 and that Cdc15 and Tem1 function in a complex to direct the exit from mitosis.

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γ-Tubulin Plays an Essential Role in the Coordination of Mitotic Events

Molecular Biology of the Cell ◽

10.1091/mbc.e03-06-0405 ◽

2004 ◽

Vol 15 (3) ◽

pp. 1374-1386 ◽

Cited By ~ 45

Author(s):

Natalie L. Prigozhina ◽

C. Elizabeth Oakley ◽

Amanda M. Lewis ◽

Tania Nayak ◽

Stephen A. Osmani ◽

...

Keyword(s):

Mitotic Spindle ◽

Essential Role ◽

Time Lapse ◽

Mitotic Checkpoint ◽

Microtubule Assembly ◽

Cycle Period ◽

Restrictive Temperature ◽

Checkpoint Control ◽

Sensitive Allele

Recent data from multiple organisms indicate that γ-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the γ-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (∼6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that γ-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.

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Genes Involved in Sister Chromatid Separation and Segregation in the Budding Yeast Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/159.2.453 ◽

2001 ◽

Vol 159 (2) ◽

pp. 453-470

Author(s):

Sue Biggins ◽

Needhi Bhalla ◽

Amy Chang ◽

Dana L Smith ◽

Andrew W Murray

Keyword(s):

Chromosome Segregation ◽

Sister Chromatid ◽

Mitotic Spindle ◽

Budding Yeast ◽

Temperature Sensitive ◽

Chromosome Behavior ◽

Yeast Saccharomyces Cerevisiae ◽

Sister Chromatids ◽

Marked Chromosome ◽

Sister Chromatid Separation

Abstract Accurate chromosome segregation requires the precise coordination of events during the cell cycle. Replicated sister chromatids are held together while they are properly attached to and aligned by the mitotic spindle at metaphase. At anaphase, the links between sisters must be promptly dissolved to allow the mitotic spindle to rapidly separate them to opposite poles. To isolate genes involved in chromosome behavior during mitosis, we microscopically screened a temperature-sensitive collection of budding yeast mutants that contain a GFP-marked chromosome. Nine LOC (loss of cohesion) complementation groups that do not segregate sister chromatids at anaphase were identified. We cloned the corresponding genes and performed secondary tests to determine their function in chromosome behavior. We determined that three LOC genes, PDS1, ESP1, and YCS4, are required for sister chromatid separation and three other LOC genes, CSE4, IPL1, and SMT3, are required for chromosome segregation. We isolated alleles of two genes involved in splicing, PRP16 and PRP19, which impair α-tubulin synthesis thus preventing spindle assembly, as well as an allele of CDC7 that is defective in DNA replication. We also report an initial characterization of phenotypes associated with the SMT3/SUMO gene and the isolation of WSS1, a high-copy smt3 suppressor.

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The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

Canadian Journal of Biochemistry ◽

10.1139/o73-214 ◽

1973 ◽

Vol 51 (12) ◽

pp. 1588-1597 ◽

Cited By ~ 7

Author(s):

David T. Denhardt ◽

Makoto Iwaya ◽

Grant McFadden ◽

Gerald Schochetman

Keyword(s):

Dna Polymerase ◽

Dna Polymerases ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Dna Polymerase Iii ◽

Single Stranded Dna ◽

E Coli ◽

Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

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The Mitochondrial Nucleoid Protein, Mgm101p, of Saccharomyces cerevisiae Is Involved in the Maintenance of ρ+ and ori/rep-Devoid Petite Genomes but Is Not Required for Hypersuppressive ρ− mtDNA

Genetics ◽

10.1093/genetics/160.4.1389 ◽

2002 ◽

Vol 160 (4) ◽

pp. 1389-1400

Author(s):

Xiao Ming Zuo ◽

G Desmond Clark-Walker ◽

Xin Jie Chen

Keyword(s):

Saccharomyces Cerevisiae ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mitochondrial Rna ◽

Mtdna Copy Number ◽

Mitochondrial Rna Polymerase ◽

Mitochondrial Nucleoids ◽

Mtdna Replication ◽

Sensitive Allele ◽

Discriminatory Effect

Abstract The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional ρ+ genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a ρ+ strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have ρ− genomes that are stably maintained. Interestingly, all surviving ρ− mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS ρ− mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of ρ+ and ori/rep-devoid ρ− mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS ρ− genome is stably maintained in a mgm101, rpo41 double mutant.

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Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽

10.1093/genetics/148.3.989 ◽

1998 ◽

Vol 148 (3) ◽

pp. 989-1005 ◽

Cited By ~ 5

Author(s):

Keiko Umezu ◽

Neal Sugawara ◽

Clark Chen ◽

James E Haber ◽

Richard D Kolodner

Keyword(s):

Dna Replication ◽

Dna Binding ◽

Protein A ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Physical Analysis ◽

Single Stranded Dna ◽

Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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Casein kinase I-like protein kinases encoded by YCK1 and YCK2 are required for yeast morphogenesis.

Molecular and Cellular Biology ◽

10.1128/mcb.13.5.2870 ◽

1993 ◽

Vol 13 (5) ◽

pp. 2870-2881 ◽

Cited By ~ 83

Author(s):

L C Robinson ◽

M M Menold ◽

S Garrett ◽

M R Culbertson

Keyword(s):

Protein Kinase ◽

Eukaryotic Cell ◽

Casein Kinase ◽

Temperature Sensitive ◽

Protein Kinase Activity ◽

Restrictive Temperature ◽

Loss Of Function ◽

Casein Kinase I ◽

Yeast Saccharomyces Cerevisiae

Casein kinase I is an acidotropic protein kinase class that is widely distributed among eukaryotic cell types. In the yeast Saccharomyces cerevisiae, the casein kinase I isoform encoded by the gene pair YCK1 and YCK2 is a 60- to 62-kDa membrane-associated form. The Yck proteins perform functions essential for growth and division; either alone supports growth, but loss of function of both is lethal. We report here that casein kinase I-like activity is associated with a soluble Yck2-beta-galactosidase fusion protein in vitro and that thermolabile protein kinase activity is exhibited by a protein encoded by fusion of a temperature-sensitive yck2 allele with lacZ. Cells carrying the yck2-2ts allele arrest at restrictive temperature with multiple, elongated buds containing multiple nuclei. This phenotype suggests that the essential functions of the Yck proteins include roles in bud morphogenesis, possibly in control of cell growth polarity, and in cytokinesis or cell separation. Further, a genetic relationship between the yck2ts allele and deletion of CDC55 indicates that the function of Yck phosphorylation may be related to that of protein phosphatase 2A activity.

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CDC68, a yeast gene that affects regulation of cell proliferation and transcription, encodes a protein with a highly acidic carboxyl terminus

Molecular and Cellular Biology ◽

10.1128/mcb.11.11.5718-5726.1991 ◽

1991 ◽

Vol 11 (11) ◽

pp. 5718-5726

Author(s):

A Rowley ◽

R A Singer ◽

G C Johnston

Keyword(s):

Gene Expression ◽

Cell Cycle ◽

Cycle Performance ◽

Carboxyl Terminus ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Negative Effects ◽

Yeast Saccharomyces Cerevisiae ◽

Acid Protein ◽

Mutant Cells

The cell cycle of the budding yeast Saccharomyces cerevisiae has been investigated through the study of conditional cdc mutations that specifically affect cell cycle performance. Cells bearing the cdc68-1 mutation (J. A. Prendergast, L. E. Murray, A. Rowley, D. R. Carruthers, R. A. Singer, and G. C. Johnston, Genetics 124:81-90, 1990) are temperature sensitive for the performance of the G1 regulatory event, START. Here we describe the CDC68 gene and present evidence that the CDC68 gene product functions in transcription. CDC68 encodes a 1,035-amino-acid protein with a highly acidic and serine-rich carboxyl terminus. The abundance of transcripts from several unrelated genes is decreased in cdc68-1 mutant cells after transfer to the restrictive temperature, while at least one transcript, from the HSP82 gene, persists in an aberrant fashion. Thus, the cdc68-1 mutation has both positive and negative effects on gene expression. Our findings complement those of Malone et al. (E. A. Malone, C. D. Clark, A. Chiang, and F. Winston, Mol. Cell. Biol. 11:5710-5717, 1991), who have independently identified the CDC68 gene (as SPT16) as a transcriptional suppressor of delta-insertion mutations. Among transcripts that rapidly become depleted in cdc68-1 mutant cells are those of the G1 cyclin genes CLN1, CLN2, and CLN3/WHI1/DAF1, whose activity has been previously shown to be required for the performance of START. The decreased abundance of cyclin transcripts in cdc68-1 mutant cells, coupled with the suppression of cdc68-1-mediated START arrest by the CLN2-1 hyperactive allele of CLN2, shows that the CDC68 gene affects START through cyclin gene expression.

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