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Susan M Gasser

Publications and source records attributed to Susan M Gasser.

13 recordsLinked to original sources
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↗
PubMed2002

Intracellular trafficking of yeast telomerase components.

Telomerase uses an internal RNA moiety as template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the telomerase holoenzyme contains the telomerase reverse transcriptase subunit Est2p, the telomerase RNA moiety TLC1, the telomerase associated proteins Est1p and Est3p, and Sm proteins. Here we assess telomerase assembly by determining the localization of telomerase components. We found that Est1p, Est2p and TLC1 can migrate independently of each other to the nucleus. With limiting amounts of TLC1, overexpressed Est1p and Est2p accumulated in the nucleolus, whereas enzymatically active Est2p-TLC1 complexes are distributed over the entire nucleus. The distribution to the nucleoplasm depended on the specific interaction between Est2p and TLC1 but was independent of Est1p and Est3p. Altogether, our results suggest a role of the nucleolus in telomerase biogenesis. We also describe experiments that support a transient cytoplasmic localization of TLC1 RNA.

Active Transport, Cell Nucleus↗
PubMedDate not supplied

Myosin-like proteins 1 and 2 are not required for silencing or telomere anchoring, but act in the Tel1 pathway of telomere length control.

The positioning of chromosomal domains in interphase nuclei is thought to facilitate transcriptional repression in yeast. It has been reported that two large coiled-coil proteins of the nuclear envelope, myosin-like proteins 1 and 2, play direct roles in anchoring yeast telomeres to the nuclear periphery, thereby creating a subcompartment enriched for Sir proteins. We have created strains containing complete deletions of mlp1 and mlp2 genes, as well as the double null strain, and find no evidence for the disruption of telomere anchoring at the nuclear periphery in these cells. We also detect no disruption of telomere-associated gene silencing. We confirm, on the other hand, that mlp mutants are particularly sensitive to DNA-damaging agents, such as bleomycin. Moreover, we show that rather than having short telomeres as in yKu-deficient strains, the mlp1 mlp2 strains have extended telomeres, resembling phenotypes of mutations in rif1. Whereas the mlp1 mlp2 mutations act on a pathway of telomere length regulation different from that of yKu70, the effects of the tel1 deletion are epistatic to the mlp mutations, suggesting that the Mlp proteins restrict telomere length in wild-type cells by influencing the Rif-Tel1 pathway of telomerase regulation.

Bleomycin↗