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Susan M Gasser

Publications and source records attributed to Susan M Gasser.

At least 37 records · Page 2Linked to original sources
PubMed2004

Long-range compaction and flexibility of interphase chromatin in budding yeast analyzed by high-resolution imaging techniques.

Little is known about how chromatin folds in its native state. Using optimized in situ hybridization and live imaging techniques have determined compaction ratios and fiber flexibility for interphase chromatin in budding yeast. Unlike previous studies, ours examines nonrepetitive chromatin at intervals short enough to be meaningful for yeast chromosomes and functional domains in higher eukaryotes. We reconcile high-resolution fluorescence in situ hybridization data from intervals of 14-100 kb along single chromatids with measurements of whole chromosome arms (122-623 kb in length), monitored in intact cells through the targeted binding of bacterial repressors fused to GFP derivatives. The results are interpreted with a flexible polymer model and suggest that interphase chromatin exists in a compact higher-order conformation with a persistence length of 170-220 nm and a mass density of approximately 110-150 bp/nm. These values are equivalent to 7-10 nucleosomes per 11-nm turn within a 30-nm-like fiber structure. Comparison of long and short chromatid arm measurements demonstrates that chromatin fiber extension is also influenced by nuclear geometry. The observation of this surprisingly compact chromatin structure for transcriptionally competent chromatin in living yeast cells suggests that the passage of RNA polymerase II requires a very transient unfolding of higher-order chromatin structure.

Chromatin↗
PubMed2004

Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins.

In budding yeast, the nuclear periphery forms a subcompartment in which telomeres cluster and SIR proteins concentrate. To identify the proteins that mediate chromatin anchorage to the nuclear envelope, candidates were fused to LexA and targeted to an internal GFP-tagged chromosomal locus. Their ability to shift the locus from a random to a peripheral subnuclear position was monitored in living cells. Using fusions that cannot silence, we identify YKu80 and a 312-aa domain of Sir4 (Sir4(PAD)) as minimal anchoring elements, each able to relocalize an internal chromosomal locus to the nuclear periphery. Sir4(PAD)-mediated tethering requires either the Ku complex or Esc1, an acidic protein that is localized to the inner face of the nuclear envelope even in the absence of Ku, Sir4 or Nup133. Finally, we demonstrate that Ku- and Esc1-dependent pathways mediate natural telomere anchoring in vivo. These data provide the first unambiguous identification of protein interactions that are both necessary and sufficient to localize chromatin to the nuclear envelope.

Alleles↗
PubMed2004

Redundancy, insult-specific sensors and thresholds: unlocking the S-phase checkpoint response.

DNA damage that is not properly repaired during genomic replication is a major source of gross chromosomal rearrangements and sequence loss during cell proliferation. In higher eukaryotes such mutations increase the risk of cancer. Eukaryotic cells have multiple checkpoint responses activated by DNA damage and stalled replication forks. We focus here on fork-associated events that activate and respond to S-phase checkpoint kinases.

Animals↗
PubMed2004

The function of nuclear architecture: a genetic approach.

Eukaryotic genomes are distributed on linear chromosomes that are grouped together in the nucleus, an organelle separated from the cytoplasm by a characteristic double membrane studded with large proteinaceous pores. The chromatin within chromosomes has an as yet poorly characterized higher-order structure, but in addition to this, chromosomes and specific subchromosomal domains are nonrandomly positioned in nuclei. This review examines functional implications of the long-range organization of the genome in interphase nuclei. A rigorous test of the physiological importance of nuclear architecture is achieved by introducing mutations that compromise both structure and function. Focussing on such genetic approaches, we address general concepts of interphase nuclear order, the role of the nuclear envelope (NE) and lamins, and finally the importance of spatial organization for DNA replication and heritable gene expression.

Animals↗
PubMed2003

Modulation of drug sensitivity in yeast cells by the ATP-binding domain of human DNA topoisomerase IIalpha.

Epipodophyllotoxins are effective antitumour drugs that trap eukaryotic DNA topoisomerase II in a covalent complex with DNA. Based on DNA cleavage assays, the mode of interaction of these drugs was proposed to involve amino acid residues of the catalytic site. An in vitro binding study, however, revealed two potential binding sites for etoposide within human DNA topoisomerase IIalpha (htopoIIalpha), one in the catalytic core of the enzyme and one in the ATP-binding N-terminal domain. Here we have tested how N-terminal mutations that reduce the affinity of the site for etoposide or ATP affect the sensitivity of yeast cells to etoposide. Surprisingly, when introduced into full-length enzymes, mutations that lower the drug binding capacity of the N-terminal domain in vitro render yeast more sensitive to epipodophyllotoxins. Consistently, when the htopoIIalpha N-terminal domain alone is overexpressed in the presence of yeast topoII, cells become more resistant to etoposide. Point mutations that weaken etoposide binding eliminate this resistance phenotype. We argue that the N-terminal ATP-binding pocket competes with the active site of the holoenzyme for binding etoposide both in cis and in trans with different outcomes, suggesting that each topoisomerase II monomer has two non-equivalent drug-binding sites.

Adenosine Triphosphate↗
PubMed2003

DNA polymerase stabilization at stalled replication forks requires Mec1 and the RecQ helicase Sgs1.

To ensure proper replication and segregation of the genome, eukaryotic cells have evolved surveillance systems that monitor and react to impaired replication fork progression. In budding yeast, the intra-S phase checkpoint responds to stalled replication forks by downregulating late-firing origins, preventing spindle elongation and allowing efficient resumption of DNA synthesis after recovery from stress. Mutations in this pathway lead to high levels of genomic instability, particularly in the presence of DNA damage. Here we demonstrate by chromatin immunoprecipitation that when yeast replication forks stall due to hydroxyurea (HU) treatment, DNA polymerases alpha and epsilon are stabilized for 40-60 min. This requires the activities of Sgs1, a member of the RecQ family of DNA helicases, and the ATM-related kinase Mec1, but not Rad53 activation. A model is proposed whereby Sgs1 helicase resolves aberrantly paired structures at stalled forks to maintain single-stranded DNA that allows RP-A and Mec1 to promote DNA polymerase association.

DNA Helicases↗
PubMed2003

RecQ helicases: multiple roles in genome maintenance.

The RecQ helicases are highly conserved in evolution and are required for maintaining genome stability in all organisms. In humans, loss of RecQ helicase function is associated with predisposition to cancer and/or premature ageing. Recent data show that RecQ helicases have several roles during S phase of the cell cycle, ranging from facilitating the resumption of DNA synthesis at sites of replication fork breakdown to resolving structures during the process of homologous recombination.

Animals↗
PubMed2002

Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast.

BACKGROUND: The positioning of chromosomal domains within interphase nuclei is thought to facilitate transcriptional repression in yeast. Although this is particularly well characterized for telomeres, the molecular basis of their specific subnuclear organization is poorly understood. The use of live fluorescence imaging overcomes limitations of in situ staining on fixed cells and permits the analysis of chromatin dynamics in relation to stages of the cell cycle. RESULTS: We have characterized the dynamics of yeast telomeres and their associated domains of silent chromatin by using rapid time-lapse microscopy. In interphase, native telomeres are highly dynamic but remain within a restricted volume adjacent to the nuclear envelope. This constraint is lost during mitosis. A quantitative analysis of selected mutants shows that the yKu complex is necessary for anchoring some telomeres at the nuclear envelope (NE), whereas the myosin-like proteins Mlp1 and Mlp2 are not. We are able to correlate increased telomeric repression with increased anchoring and show that silent chromatin is tethered to the NE in a Sir-dependent manner in the absence of the yKu complex. Sir-mediated anchoring is S phase specific, while the yKu-mediated pathway functions throughout interphase. Subtelomeric elements of yeast telomere structure influence the relative importance of the yKu- and Sir-dependent mechanisms. CONCLUSIONS: Interphase positioning of telomeres can be achieved through two partially redundant mechanisms. One requires the heterodimeric yKu complex, but not Mlp1 and Mlp2. The second requires Silent information regulators, correlates with transcriptional repression, and is specific to S phase.

Cell Nucleus↗
PubMed2002

ORC and the intra-S-phase checkpoint: a threshold regulates Rad53p activation in S phase.

The intra-S-phase checkpoint in yeast responds to stalled replication forks by activating the ATM-like kinase Mec1 and the CHK2-related kinase Rad53, which in turn inhibit spindle elongation and late origin firing and lead to a stabilization of DNA polymerases at arrested forks. A mutation that destabilizes the second subunit of the Origin Recognition Complex, orc2-1, reduces the number of functional replication forks by 30% and severely compromises the activation of Rad53 by replication stress or DNA damage in S phase. We show that the restoration of the checkpoint response correlates in a dose-dependent manner with the restoration of pre-replication complex formation in G1. Other forms of DNA damage can compensate for the reduced level of fork-dependent signal in the orc2-1 mutant, yet even in wild-type cells, the amount of damage required for Rad53 activation is higher in S phase than in G2. Our data suggest the existence of an S-phase-specific threshold that may be necessary to allow cells to tolerate damage-like DNA structures present at normal replication forks.

Cell Cycle Proteins↗
PubMed2002

An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing.

The Dbf4Cdc7 kinase acts at the level of individual origins to promote the initiation of DNA replication. We demonstrate through both immunoprecipitation and two-hybrid assays that a domain comprising the first 296 aa of Dbf4p interacts with Orc2p and Orc3p subunits of the origin recognition complex (ORC). Given that the activation of Rad53 kinase in response to the DNA replication checkpoint leads to the release of Dbf4p from an ORC-containing chromatin fraction, we also examined interaction between Dbf4p and Rad53p. This same domain of Dbf4p binds specifically to the forkhead homology-associated (FHA) domains of Rad53p. Cell cycle arrest in G(2)M, provoked by the overexpression of the Dbf4 domain, is suppressed in a rad53 mutant. Moreover, its overexpression perturbs the regulation of late, but not early, origin firing in wild-type cells after treatment with hydroxyurea.

Binding Sites↗
PubMed2002

RecQ helicases: at the heart of genetic stability.

The checkpoint-mediated control of DNA replication is essential for maintaining the stability of the genome and preventing cancer in humans. The RecQ family of helicases has been shown to be important for the maintenance of genomic integrity in organisms ranging from bacteria to man. We propose that the RecQ homologue, Sgs1p, has an important function in the S-phase checkpoint response of budding yeast, where it may be both a 'sensor' for damage during replication and a 'resolvase' for structures that arise at paused forks. RecQ helicases may serve a unique function that integrates checkpoint proteins with the recombination and replication fork machinery.

Adenosine Triphosphatases↗
PubMed2002

RecQ helicases and genome stability: lessons from model organisms and human disease.

Maintaining the integrity of genetic information is fundamental for the life of a cell and the survival of a species. Cells can encounter DNA damage as a consequence of normal cellular metabolism or as a result of exposure to chemical or physical agents. Eukaryotic cells have developed a network of responses in order to deal with DNA damage thereby preserving the integrity of their genetic information. In the presence of extensive genetic insult, a surveillance mechanism or "checkpoint" is activated. The activation of this signal transduction pathway leads to an arrest of cell cycle progression to prevent replication and segregation of damaged DNA molecules and to induce transcription of several repair genes. Existing repair mechanisms are also mobilised, in a coordinated effort to restore the original DNA structure. Genes involved in either cell cycle checkpoints, DNA repair or genes that maintain the fidelity of chromosome segregation are often termed "antimutators" or "caretaker" genes, because they control the stability of the genome and prevent accumulation of mutations in so-called "gatekeeper" genes. This latter group of genes directly regulate the growth of tumours either by inhibiting growth or promoting death. A fundamental requirement for many DNA metabolism processes is the separation of the complementary strands of the DNA duplex. This is promoted by DNA helicases, which unwind nucleic-acid duplexes in an ATP-dependent manner to provide access to the template for proteins of the replication, recombination, repair and transcription machineries. Multiple DNA helicase families have been identified, all containing seven hallmark helicase motifs; members within each helicase family also share sequence homologies beyond and between these motifs. One example is the RecQ helicase family, named after the RecQ protein of Escherichia coli, which was identified during a search for mutants sensitive to thymine starvation. Five members of the RecQ family have been identified in the human genome, and mutations in three of the genes are responsible for genetic diseases that are characterised by genomic instability and a high incidence of cancer. Because mutants in RecQ family genes in other species also have unstable chromosomes, it was proposed that members of the RecQ helicase family play a central role in the maintenance of genomic stability and thereby the prevention of tumorigenesis.

Adenosine Triphosphatases↗
PubMed2002

Visualizing chromatin dynamics in interphase nuclei.

Real-time fluorescence microscopy has emerged as a powerful tool for examining chromatin dynamics. The initial lesson is that much of the genome, particularly in yeast, is highly dynamic. Its movement within the interphase nucleus is correlated with metabolic activity. Nonetheless, the nucleus is an organelle with conserved rules of organization. Determining the distribution and regulation of mobile domains in interphase chromosomes, and characterizing sites of anchorage, will undoubtedly shed new light on the function of nuclear order.

Animals↗