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S M Gasser

Publications and source records attributed to S M Gasser.

At least 73 records · Page 4Linked to original sources
PubMed1995

The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing.

The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.

Cell Compartmentation↗
PubMed1994

RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomere-telomere interactions.

Repressor Activator Protein 1 (RAP1) of Saccharomyces cerevisiae is an abundant nuclear protein implicated in telomere length maintenance, transactivation, and in the establishment of silent chromatin domains. The RAP1 binding site 5' of the yeast HIS4 gene is also a region of hyperrecombination in meiosis. We report here that as RAP1 binds its recognition consensus, it appears to untwist double-stranded DNA, which we detect as the introduction of a negative supercoil in circularization assays. Coincident with the RAP1-dependent untwisting, we observe stimulation of the association of a single-stranded yeast telomeric sequence with its homologous double-stranded sequence in a supercoiled plasmid. This unusual distortion of the DNA double helix by RAP1 may contribute to the RAP1-dependent enhancement of recombination rates and promote non-duplex strand interactions at telomeres.

Base Sequence↗
PubMed1994

Phosphorylation of the C-terminal domain of yeast topoisomerase II by casein kinase II affects DNA-protein interaction.

Eukaryotic DNA topoisomerase II is an abundant nuclear enzyme that is essential for cell proliferation. This homodimeric enzyme catalyzes the cleavage and re-ligation of double-stranded DNA required to separate replicated sister chromatids. Both biochemical and genetic studies show that its catalytic activity is required for chromosome condensation and segregation, and that its decatenation activity can be stimulated by a variety of protein kinases in vitro. In budding yeast, topoisomerase II is most highly phosphorylated in metaphase, and casein kinase II (CKII) was shown to be the major kinase modifying topoisomerase II. We have investigated the effects of phosphorylation of yeast topoisomerase II by CKII in vitro, by means of gel-retardation and filter binding assays. The phosphorylation of the C terminus of topoisomerase II by CKII appears to increase the stability of the complex formed with linear DNA fragments, while dephosphorylation has the opposite effect. Rephosphorylation of phosphatase-treated topoisomerase II by chicken casein kinase II restores a stable protein-DNA complex using a linear DNA fragment. The enhanced stability of the topoisomerase II-DNA complex is also observed with relaxed circular DNA, but not with supercoiled minicircles, in agreement with published results using topoisomerase II from Drosophila. Limited proteolysis and probing with domain-specific antibodies shows that, with the exception of a weakly modified residue between amino acid residues 660 and 1250, all residues modified by casein kinase II are in the last 180 amino acid residues of yeast topoisomerase II.

Binding Sites↗
PubMed1994

The yeast protein encoded by PUB1 binds T-rich single stranded DNA.

We have characterized binding activities in yeast which recognise the T-rich strand of the yeast ARS consensus element and have purified two of these to homogeneity. One (ACBP-60) is detectable in both nuclear and whole cell extracts, while the other (ACBP-67) is apparent only after fractionation of extracts by heparin-sepharose chromatography. The major binding activity detected in nuclear extracts was purified on a sequence-specific DNA affinity column as a single polypeptide with apparent mobility of 60kDa (ACBP-60). This protein co-fractionates with nuclei, is present at several thousand copies per cell and has a Kd for the T-rich single strand of the ARS consensus between 10(-9) and 10(-10) M. Competition studies with simple nucleic acid polymers show that ACBP-60 has marginally higher affinity for poly dT30 than for a 30 nt oligomer containing the T-rich strand of ARS 307, and approximately 10 fold higher affinity for poly rU. Internal sequence information of purified p60 reveals identity with the open reading frames of genes PUB1 and RNP1 which encode polyuridylate binding protein(s). The second binding activity, ACBP-67, also binds specifically to the T-rich single strand of the ARS consensus, but with considerably lower affinity than ACBP-60. Peptide sequence reveals that the 67kDa protein is identical to the major polyA binding protein in yeast, PAB1.

Base Composition↗
PubMed1994

Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain.

We present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.

Calcium↗
PubMed1994

The regulation of DNA topoisomerase II by casein kinase II.

DNA topoisomerase II is an essential nuclear enzyme required for the proper condensation and segregation of chromosomes during mitotic and meiotic cell division. The enzyme exists in the cell as a phosphoprotein, and it is most highly phosphorylated in G2 and M-phases of the cell cycle. We have shown that topoisomerase II is the target of casein kinase II (CKII) in yeast by comparison of in vivo and in vitro phosphotryptic peptide maps. Limited proteolysis and probing with domain specific antibodies show that with the exception of a weakly modified residue between aa 660 and aa 1250, all residues modified by CKII are in the last 200 amino acids of yeast topoisomerase II. This C-terminal domain is the least conserved region of the enzyme and truncation of the enzyme shows that it is nonessential for activity in vitro. However, the fully dephosphorylated full-size protein is nearly inactive in decatenation assays, and activity can be restored by phosphorylation by CKII. To reconcile these observations, we propose that the C-terminal region is a negative regulatory domain, counteracted by phosphorylation within the domain itself. To test this hypothesis we have mutagenised 12 potential CKII phosphoacceptor sites in the C-terminus of topoisomerase II and introduced the mutant genes into a yeast strain which has a temperature sensitive top2 gene. The growth of the transformed strains is monitored at nonpermissive temperature to determine whether C-terminal phosphorylation is important for mitotic growth. In addition, we have purified the mutant enzymes to homogeneity for in vitro assays.

Amino Acid Sequence↗
PubMed1993

SIR3 and SIR4 proteins are required for the positioning and integrity of yeast telomeres.

Heritable inactivation of genes occurs in specific chromosomal domains located at the silent mating type loci and at telomeres of S. cerevisiae. The SIR genes (for silent information regulators) are trans-acting factors required for this repression mechanism. We show here that the SIR3 and SIR4 gene products have a sub-nuclear localization similar to the telomere-associated RAP1 protein, which is found primarily in foci at the nuclear periphery of fixed yeast spheroplasts. In strains deficient for either SIR3 or SIR4, telomeres lose their perinuclear localization, as monitored by RAP1 immunofluorescence. The length of the telomeric repeat shortens in sir3 and sir4 mutant strains, and the mitotic stability of chromosome V is reduced. These data suggest that SIR3 and SIR4 are required for both the integrity and subnuclear localization of yeast telomeres, the loss of which correlates with loss of telomere-associated gene repression.

Cell Compartmentation↗
PubMed1993

Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites.

Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats. RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo. We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting. Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY. Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides. In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex. The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp. This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes.

Amino Acid Sequence↗
PubMed1993

Telomeres and the functional architecture of the nucleus.

The single molecule of DNA that constitutes a eukaryotic chromosome begins and ends with a stretch of repetitive DNA known as a telomere. These sequences appear to be necessary to preserve the integrity of the genetic material through the cell cycle. Telomeric DNA is organized into regions of non-nucleosomal chromatin called the telosome, which can interact with other telosomes and with the nuclear envelope. This review focuses on cytological evidence for these interactions and on recent insights into the molecular organization of the telomeric complex.

Journal Article↗
PubMed1993

Casein kinase II copurifies with yeast DNA topoisomerase II and re-activates the dephosphorylated enzyme.

Mitotic division in yeast requires the activity of topoisomerase II, a DNA topology modifying enzyme that is able to disentangle sister chromatids after DNA replication. Previous work has shown that topoisomerase II is a phosphoprotein in intact yeast cells. We show here that when dephosphorylated in vitro, topoisomerase II is unable to cleave or decatenate kinetoplast DNA. An efficient kinase activity that modifies topoisomerase II on seven major sites was found to copurify with the enzyme purified from yeast. Characterization of this kinase, analysis of phosphotryptic peptides, and studies with a yeast mutant deficient in casein kinase II, indicate that the copurifying kinase is casein kinase II (CKII). Topoisomerase II itself has no self-phosphorylating activity. Modification of topoisomerase II by the copurifying kinase is sufficient to restore decatenation activity after dephosphorylation by alkaline phosphatase. The CKII target sites have been mapped to multiple serine and threonine residues on 4 tryptic fragments within the C-terminal 350 amino acids of yeast topoisomerase II. These results are consistent with a model in which the C-terminal domain of topoisomerase II is a negative regulatory domain that is neutralized by phosphorylation.

Amino Acid Sequence↗
PubMed1992

Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo.

The decatenation activity of DNA topoisomerase II is essential for viability as eukaryotic cells traverse mitosis. Phosphorylation has been shown to stimulate topoisomerase II activity in vitro. Here we show that topoisomerase II is a phosphoprotein in yeast and that the level of incorporated phosphate is significantly higher at mitosis than in G1. Comparison of tryptic phosphopeptide maps reveals that the major phosphorylation sites in vivo are targets for casein kinase II. Incorporation of phosphate into topoisomerase II is nearly undetectable at the non-permissive temperature in a conditional casein kinase II mutant. The sites modified by casein kinase II are located in the extreme C-terminal domain of topoisomerase II. This domain is absent in prokaryotic and highly divergent among eukaryotic type II topoisomerases, and may serve to regulate functions of topoisomerase II that are unique to eukaryotic cells.

Amino Acids↗
PubMed1992

Topoisomerase II: its functions and phosphorylation.

The gene encoding topoisomerase II in yeast is unique and essential, required for both mitotic and meiotic proliferation. The use of temperature-sensitive mutants in topoisomerase II have demonstrated roles in the relaxation of tortional stress, reduction of recombination rates, and in the separation of sister chromatids after replication. In vertebrate cells, topoisomerase II was shown to be the most abundant component of the metaphase chromosomal scaffold, and has been shown to play a role in chromosome condensation in vitro. The cell cycle control of chromosome condensation may well require phosphorylation of topoisomerase II, since the enzyme is more highly phosphorylated in metaphase than in G1. Recent studies have identified casein kinase II as the major enzyme phosphorylating topoisomerase II in intact yeast cells. The target sites of CKII are exclusively in the C-terminal 400 amino acids of topoisomerase II, the region that is most divergent among the eukaryotic type II enzymes and which is absent in the bacterial gyrase homologues.

Casein Kinase II↗
PubMed1992

Localization of RAP1 and topoisomerase II in nuclei and meiotic chromosomes of yeast.

Topoisomerase II (topoII) and RAP1 (Repressor Activator Protein 1) are two abundant nuclear proteins with proposed structural roles in the higher-order organization of chromosomes. Both proteins co-fractionate as components of nuclear scaffolds from vegetatively growing yeast cells, and both proteins are present as components of pachytene chromosome, co-fractionating with an insoluble subfraction of meiotic nuclei. Immunolocalization using antibodies specific for topoII shows staining of an axial core of the yeast meiotic chromosome, extending the length of the synaptonemal complex. RAP1, on the other hand, is located at the ends of the paired bivalent chromosomes, consistent with its ability to bind telomeric sequences in vitro. In interphase nuclei, again in contrast to anti-topoII, anti-RAP1 gives a distinctly punctate staining that is located primarily at the nuclear periphery. Approximately 16 brightly staining foci can be identified in a diploid nucleus stained with anti-RAP1 antibodies, suggesting that telomeres are grouped together, perhaps through interaction with the nuclear envelope.

Base Sequence↗
PubMed1992

DNA loops: structural and functional properties of scaffold-attached regions.

The long DNA molecules of eukaryotic genomes appear to be organized into large loops formed by the binding of dispersed DNA sequences to non-histone proteins. This partitioning of DNA into topologically constrained units constitutes one of the highest orders of DNA packing in chromosomes. DNA loops are likely to define functional units as well as topological domains, contributing to the regulation of gene expression and DNA replication. This review presents recent work on the properties of the DNA sequences and proteins thought to be involved in loop formation, and on their possible significance for replication and transcription.

Base Sequence↗
PubMed1991

Identification and purification of a protein that binds the yeast ARS consensus sequence.

We have identified a yeast protein that binds specifically to the ARS consensus sequence. By two-step chromatography we have purified the factor to apparent homogeneity as a single polypeptide of 67 kd. The purified ARS consensus-binding protein (ACBP) recognizes the ARS consensus of the four genomic ARS elements tested, binding preferentially to the T-rich single strand. Point mutations in the consensus significantly reduce the affinity of the single-strand binding. At the histone H4 ARS, ACBP recognizes both the perfect ARS consensus and a 9/11 match 3' of it. These two binding sites correlate with the boundaries of the minimal functional H4 ARS element. A similar configuration of binding sites is found at ARS1. We propose a model implicating this factor in an early step of the initiation of DNA replication.

Base Sequence↗