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S M Gasser

Publications and source records attributed to S M Gasser.

At least 37 records · Page 2Linked to original sources
PubMed2000

RecQ-like helicases: the DNA replication checkpoint connection.

The eukaryotic homologues of the Escherichia coli RecQ DNA helicase play conserved roles in the maintenance of genome stability. Results obtained in yeast and mammalian systems are beginning to form a coherent picture about what these helicases do to ensure normal cell division and why humans who lack these enzymes are cancer prone. Recent data suggest that the yeast enzyme Sgs1p, as well as two human homologues, which are encoded by the Bloom's and Werner's syndrome genes, function during DNA replication and possibly in a replication checkpoint specific to S phase.

Adenosine Triphosphatases↗
PubMed1999

Taz1p and Teb1p, two telobox proteins in Schizosaccharomyces pombe, recognize different telomere-related DNA sequences.

Band shift assays were used to study proteins from the fission yeast that bind double-stranded telomeric repeat sequences. We also examine general DNA binding properties of the telobox domain, which characterizes telomere-binding proteins from a range of species. We demonstrate that Taz1p has a high affinity for the fission yeast telomeric repeat, consistent with genetic results implicating this protein in telomere maintenance. A second Schizosaccharomyces pombe telobox protein, Teb1p, is shown to bind with high affinity to the vertebrate repeat and with low affinity to the fission yeast telomeric DNA. When tested on G-rich single-stranded telomeric DNA, all these proteins bind with very low affinity, much like the human telomere-binding protein TRF1. Recombinant proteins containing just the telobox domains reproduce the specificity of binding demonstrated for the corresponding full-length proteins, indicating that the telobox domain is indeed responsible for specific DNA recognition. The presence of possible Teb1p-binding sites upstream of many genes suggests a role for this protein as a general transcription factor. Finally, band shift experiments with whole cell extracts from wild-type and taz1 (-)strains suggest that in addition to Taz1p, S.pombe has another major telomere-binding activity.

Base Sequence↗
PubMed1999

A role for the Cdc7 kinase regulatory subunit Dbf4p in the formation of initiation-competent origins of replication.

Using a reconstituted DNA replication assay from yeast, we demonstrate that two kinase complexes are essential for the promotion of replication in vitro. An active Clb/Cdc28 kinase complex, or its vertebrate equivalent, is required in trans to stimulate initiation in G(1)-phase nuclei, whereas the Dbf4/Cdc7 kinase complex must be provided by the template nuclei themselves. The regulatory subunit of Cdc7p, Dbf4p, accumulates during late G(1) phase, becomes chromatin associated prior to Clb/Cdc28 activation, and assumes a punctate pattern of localization that is similar to, and dependent on, the origin recognition complex (ORC). The association of Dbf4p with a detergent-insoluble chromatin fraction in G(1)-phase nuclei requires ORC but not Cdc6p or Clb/Cdc28 kinase activity, and correlates with competence for initiation. We propose a model in which Dbf4p targets Cdc7p to the prereplication complex prior to the G(1)/S transition, by a pathway parallel to, but independent of, the Cdc6p-dependent recruitment of MCMs.

CDC28 Protein Kinase, S cerevisiae↗
PubMed1999

The cytochrome b5 tail anchors and stabilizes subdomains of human DNA topoisomerase II alpha in the cytoplasm of retrovirally infected mammalian cells.

DNA topoisomerase II (topo II) is the target of many anticancer drugs and is often altered in drug-resistant cell lines. In some tumor cell lines truncated isoforms of topo IIalpha are localized to the cytoplasm. To study the localization and function of individual enzyme domains, we have epitope-tagged several fragments of human topo IIalpha and expressed them by retroviral infection of rodent and human cells. We find that fusion of the topo II fragments to the hydrophobic tail of human liver cytochrome b5 anchors the fusion protein to the outer face of cytoplasmic membranes, as determined by colocalization with calnexin and selective detergent permeabilization. Moreover, whereas the minimal ATPase domain (aa 1-266) is weakly and diffusely expressed, addition of the cytb5 anchor (1-266-b5) increases its steady-state level 16-fold with no apparent toxicity. Similar results are obtained with the complete ATPase domain (aa 1-426). A C-terminal domain (aa 1030-1504) of human topo IIalpha containing an intact dimerization motif is stably expressed and accumulates in the nucleus. Fusion to the cytb5 anchor counteracts the nuclear localization signal and relocalizes the protein to cytoplasmic membranes. In conclusion, we describe a technique that stabilizes and targets retrovirally expressed proteins such that they are exposed on the cytoplasmic surface of cellular membranes. This approach may be of general use for regulating the nuclear accumulation of drugs or proteins in living cells.

Animals↗
PubMed1999

Relocalization of telomeric Ku and SIR proteins in response to DNA strand breaks in yeast.

Telomeric TG-rich repeats and their associated proteins protect the termini of eukaryotic chromosomes from end-to-end fusions. Associated with the cap structure at yeast telomeres is a subtelomeric domain of heterochromatin, containing the silent information regulator (SIR) complex. The Ku70/80 heterodimer (yKu) is associated both with the chromosome end and with subtelomeric chromatin. Surprisingly, both yKu and the chromatin-associated Rap1 and SIR proteins are released from telomeres in a RAD9-dependent response to DNA damage. yKu is recruited rapidly to double-strand cuts, while low levels of SIR proteins are detected near cleavage sites at later time points. Consistently, yKu- or SIR-deficient strains are hypersensitive to DNA-damaging agents. The release of yKu from telomeric chromatin may allow efficient scanning of the genome for DNA strand breaks.

Antigens, Nuclear↗
PubMed1999

In vitro DNA replication in yeast nuclear extracts.

Two assays have been developed for studying DNA replication in vitro based on nuclear extracts isolated from budding yeast cells synchronized in S phase. In the first, the template DNA for replication is provided in the form of intact yeast nuclei, usually from cells arrested in G(1). In the second assay, bacterially produced supercoiled plasmid is replicated in an S-phase nuclear extract supplemented with nucleotides and an energy-regenerating system. Semiconservative DNA replication is monitored by substitution of newly synthesized DNA with bromodeoxyuridine 5'-triphosphate (BrdUTP) and density gradient analysis. In addition, neutral-neutral two-dimensional gel analyses and, in the case of nuclei, detection of newly synthesized DNA in replication foci by DIG-dUTP incorporation can be used to monitor replication.

Cell Nucleus↗
PubMed1999

Nuclear compartments and gene regulation.

Improvements in fluorescence microscopy have allowed us to explore the three-dimensional organization of the nucleus in ways that were impossible ten years ago, revealing subdomains or compartments within the nucleus defined by their enrichments of subsets of factors. Correlations have been drawn between the silencing of a gene and its proximity to a heterochromatic compartment or to the nuclear periphery. The application of genetics and high-resolution microscopy helps examine the creation, maintenance and impact of these compartments on gene expression.

Animals↗
PubMed1999

Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts.

Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro. We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo. We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus. Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts. The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase. In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected. Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract. In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts. Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

Animals↗
PubMed1999

Mutations in the C-terminal domain of topoisomerase II affect meiotic function and interaction with the casein kinase 2 beta subunit.

Topoisomerase II is a major target of the protein kinase casein kinase 2 (PK CK2) in vivo. All major phosphorylation acceptor sites in the yeast enzyme are found in the C-terminal 350aa. The acceptor sites are generally clustered such that there is more than one modified Ser or Thr within a short peptide. Mutagenesis of the predicted acceptor sites have confirmed that five of the eight predicted sites are targeted in vitro and in vivo by PK CK2. Mutation to nonphosphorylatable, neutral residues provokes at most a 10% increase in mitotic doubling time. Truncation of the enzyme leaves the enzyme catalytically active, but slightly lengthens the doubling time during mitotic growth and impedes progress through meiosis. Since this could reflect the loss of interaction with an important ligand, we have examined whether the C-terminal domain of the yeast enzyme mediates interaction with the regulatory beta subunit of PK CK2, which was previously reported to bind topoisomerase II. We find that point mutation of the phospho-acceptor sites does not abrogate the interaction with a small region of PK CK2beta, while truncation at aa1276 or aa1236 does. The site of interaction within PK CK2beta does not coincide with the highly negatively charged spermine binding site.

Amino Acid Sequence↗
PubMed1998

Structural maintenance of chromosomes protein C-terminal domains bind preferentially to DNA with secondary structure.

Structural maintenance of chromosomes (SMC) proteins interact with DNA in chromosome condensation, sister chromatid cohesion, DNA recombination, and gene dosage compensation. How individual SMC proteins and their functional domains bind DNA has not been described. We demonstrate the ability of the C-terminal domains of Saccharomyces cerevisiae SMC1 and SMC2 proteins, representing two major subfamilies with different functions, to bind DNA in an ATP-independent manner. Three levels of DNA binding specificity were observed: 1) a >100-fold preference for double-stranded versus single-stranded DNA; 2) a high affinity for DNA fragments able to form secondary structures and for synthetic cruciform DNA molecules; and 3) a strong preference for AT-rich DNA fragments of particular types. These include fragments from the scaffold-associated regions, and an alternating poly(dA-dT)-poly(dT-dA) synthetic polymer, as opposed to a variety of other polymers. Reannealing of complementary DNA strands is also promoted primarily by the C-terminal domains. Consistent with their in vitro DNA binding activity, we show that overexpression of the SMC C termini increases plasmid loss without altering viability or cell cycle progression.

Base Composition↗
PubMed1998

Semi-conservative replication in yeast nuclear extracts requires Dna2 helicase and supercoiled template.

We describe the preparation of nuclear extracts from yeast cells synchronised in S-phase that support the aphidicolin-sensitive, semi-conservative replication of primer-free, supercoiled plasmid in vitro. This is monitored by one and two-dimensional gel electrophoresis of replication intermediates that have incorporated [alpha-32P]dATP, by the conversion of methylated template DNA into a hemi-methylated or DpnI-resistant form, and by substitution of dTTP with the heavy derivative BrdUTP, which results in a shift in density corresponding to complete second strand synthesis. We demonstrate dependence on DNA pol delta and the pol alpha/primase complex, and are able to detect putative Okazaki fragments under ATP-limiting conditions. In contrast to the semi-conservative replication of supercoiled plasmid, linear or open-circular templates incorporate labelled nucleotides through repair synthesis that produces no significant density shift on CsCl gradients. Consistent with a true replication reaction we find that semi-conservative replication of plasmid DNA is stimulated in S-phase relative to G1-phase nuclear extracts, and is independent of the recombination-promoting factor Rad52p. Using this novel system we demonstrate that semi-conservative replication, but not polymerase activity per se, requires the activity of the DNA helicase encoded by DNA2.

Adenosine Triphosphatases↗
PubMed1998

Sif2p interacts with Sir4p amino-terminal domain and antagonizes telomeric silencing in yeast.

Several regions of the Saccharomyces cerevisiae genome are subject to position-dependent transcriptional repression mediated by a multi-component nucleosome-binding complex of silent information regulator proteins (Sir2p, Sir3p and Sir4p). These proteins are present in limiting amounts in the nucleus and are targeted to specific chromosomal regions by interaction with sequence-specific DNA-binding factors. Different sites of repression compete for Sir complexes, although it is not known how Sir distribution is regulated. In a screen for factors that interact with Sir4p amino terminus, we have cloned SIF2, which encodes a WD40-repeat-containing factor that disrupts telomeric silencing when overexpressed. In contrast to deletion of SIR4, SIF2 deletion improved telomeric repression, suggesting that under normal conditions Sif2p antagonizes Sir4p function at telomeres. Sif2p overexpression altered the subnuclear localization of Sir4p, but not its protein expression level, suggesting that Sif2p may recruit Sir4p to nontelomeric sites or repression. The sif2 mutant strains were hypersensitive to a range of stress conditions, but did not have decreased viability and did not alter repression in the rDNA. In conclusion, Sif2p resembles the Sir4p regulatory proteins Sir1p and Uth4p in that it competes for the functional assembly of Sir4p at telomeres, yet unlike Sir1p or Uth4p, it does not target Sir4p to either mating-type or rDNA loci.

Binding, Competitive↗
PubMed1998

Mutation of yeast Ku genes disrupts the subnuclear organization of telomeres.

The mammalian Ku70 and Ku86 proteins form a heterodimer that binds to the ends of double-stranded DNA in vitro and is required for repair of radiation-induced strand breaks and V(D)J recombination [1,2]. Deletion of the Saccharomyces cerevisiae genes HDF1 and HDF2--encoding yKu70p and yKu80p, respectively--enhances radiation sensitivity in a rad52 background [3,4]. In addition to repair defects, the length of the TG-rich repeat on yeast telomere ends shortens dramatically [5,6]. We have shown previously that in yeast interphase nuclei, telomeres are clustered in a limited number of foci near the nuclear periphery [7], but the elements that mediate this localization remained unknown. We report here that deletion of the genes encoding yKu70p or its partner yKu80p altered the positioning of telomeric DNA in the yeast nucleus. These are the first mutants shown to affect the subnuclear localization of telomeres. Strains deficient for either yKu70p or yKu80p lost telomeric silencing, although they maintained repression at the silent mating-type loci. In addition, the telomere-associated silencing factors Sir3p and Sir4p and the TG-repeat-binding protein Rap1p lost their punctate pattern of staining and became dispersed throughout the nucleoplasm. Our results implicate the yeast Ku proteins directly in aspects of telomere organization, which in turn affects the repression of telomere-proximal genes.

Animals↗
PubMed1998

Nuclear organization and silencing: trafficking of Sir proteins.

In budding yeast genes integrated near telomeres succumb to a variegated pattern of gene repression that requires the silent information regulatory proteins Sir2p, Sir3p and Sir4p, which form a nucleosome-binding complex. Immunolocalization shows that the Sir proteins co-localize with the telomeric repeat binding protein Rap1p and with telomeric DNA in a limited number of foci near the periphery of interphase nuclei. All conditions tested so far that disrupt telomere proximal repression result in a dispersed staining pattern for Sir2p, Sir3p and Sir4p. Although the focal organization is clearly not sufficient for establishing repression, genetic studies suggest that the high local concentration of Sir proteins at telomeric foci facilitates the formation of repressed chromatin. In addition to its telomeric localization, Sir2p is shown by immunostaining and cross-linking to bind a subdomain of the nucleolus. In strains lacking an intact Sir4p, Sir3p also becomes concentrated in the nucleolus by a pathway requiring SIR2 and UTH4. This unexpected localization correlates with observed effects of sir mutations on rDNA stability and longevity, defining a new site of action for silent information regulatory factors. We report a novel WD40 repeat-containing factor, Sif2p, that binds specifically to the Sir4p N-terminus. Like Sir1p and Uth4p, Sif2p antagonizes telomeric silencing by regulating an equilibrium between alternative assembly pathways at different subnuclear loci.

Animals↗
PubMed1998

The genetics of epigenetics.

Cis-acting binding sites for transcription activators cannot explain the patterned expression of genes during development, nor a large range of phenomena that result from gene transplacement. Heritable states of transcriptional regression or activation sequences are influenced not only by the chromosomal context of the promoter but also by modifications of histones and DNA, and long-range interactions between distant chromosomal elements. The molecular dissection of these epigenetic phenomena has become an exciting topic of research, revealing highly conserved mechanisms at work in chromatin-mediated gene control.

Animals↗
PubMed1998

New systems for replicating DNA in vitro.

Current paradigms for the regulation of genomic DNA replication in eukaryotes are derived primarily from cell fusion experiments, yeast genetics, and from in vitro assays in Xenopus egg extracts. Initially, many aspects seemed irreconcilably different among the various organisms and model systems. In the past year, however, divergent approaches have arrived at a consensus on how the cell cycle regulates the initiation of DNA replication. All major players appear to be conserved from yeast to vertebrates, yet the important challenge of reconstituting eukaryotic replication from purified components remains. Three novel in vitro assays that replicate nuclear templates bring us closer to this goal.

Animals↗