Victoria Lundblad
Salk Institute For Biological Studies
Project start date: 1993-09-20
Project end date: 2013-01-31
Sponsored Links Excellgen http://Excellgen.com
TELOMERE REPLICATION AND SENESCENCE IN YEAST
Victoria Lundblad
Salk Institute For Biological Studies, La Jolla, Ca 92037-1099
Grant 5R37AG011728-17 from National Institute On Aging
Abstract: The ends of linear chromosomes, called telomeres, are essential for genomic stability and normal cellular growth. In most organisms, the primary mechanism for complete replication of telomeres relies on the enzyme telomerase. Loss of telomerase function results in a progressive decline in telomere length that heralds replicative senescence, which has been postulated to contribute to organismal aging. This enzyme is also reactivated in the majority of human tumors, indicating that telomerase is a likely target for anti-cancer therapeutics. A full appreciation of the contribution of telomere replication to these two important aspects of human biology will require a molecular understanding of how telomerase is regulated in vivo. We are using the budding yeast, S. cerevisiae, as a system for dissection of this problem, based on the assumption that what we learn in yeast will translate to human cells. Past work in our laboratory has identified three proteins, Estl, Est2 and Est3, that are subunits of yeast telomerase. The Est2 protein is the catalytic reverse transcriptase component, whereas Estl and Est3, which are dispensable for enzyme catalysis but essential in vivo for telomere replication, are positive regulatory subunits of the telomerase holoenzyme. Our genetic and biochemical analysis has shown that the Estl and Est3 subunits contribute multiple, and functionally distinct, regulatory roles to telomere replication. We have also characterized potential regulatory domains of the TLC1 RNA subunit of the yeast telomerase enzyme. Additional recent studies have led to the identification of additional genes required for regulation of telomere replication, including a potential negative regulator of telomerase. In this application, we propose a set of experiments intended to form an integrated picture of how these positive and negative regulators of telomerase control telomere replication
Keywords: Age; Aging; Binding; Binding (Molecular Function); Biochemical; Catalysis; Catalytic Core; Catalytic Domain; Catalytic Region; Catalytic Site; Catalytic Subunit; Cell Aging; Cell Senescence; Cells; Cellular Aging; Cellular Expansion; Cellular Growth; Chromosomes; Collection; Complex; DISSEC; DNA Binding; DNA Binding Interaction; Defect; Deoxynucleotide-triphosphate[{..}]DNA deoxynucleotidyltransferase (RNA-directed); Dissection; EC 2.7.7.49; Endomycetales; Enzymes; Est2 protein; Euplotes; Gene Products, RNA; GeneHomolog; Genes; Genetic; Genome Stability; Genomics; Goals; Holoenzymes; Homolog; Homologous Gene; Homologue; Human; Human Biology; Human, General; Investigation; Laboratories; Learning; Length; Man (Taxonomy); Man, Modern; Molecular; Molecular Interaction; Monitor; Nuclear; Organism; Phenotype; Play; Property; Property, LOINC Axis 2; Proteins; RNA; RNA Transcriptase; RNA, Non-Polyadenylated; RNA-Dependent DNA Polymerase; RNA-Directed DNA Polymerase; Regulation; Regulatory Protein; Reverse Transcriptase; Revertase; Ribonucleic Acid; Role; S cerevisiae; Saccharomyces cerevisiae; Saccharomycetales; Senescence; Senescence, Cellular; Senescence, Replicative; Stability, Genomic; Structure; System; System, LOINC Axis 4; TERT protein; Telomerase; Telomerase RNA Component; Translating; Translatings; Two Hybrid; Work; Yeast One Hybrid System; Yeast One/Two-Hybrid System; Yeast, Baker`s; Yeast, Brewer`s; Yeast, Budding; Yeasts; anti-cancer therapeutic; anticancer therapeutic; base; cell growth; ever shorter teleomeres protein 2; experiment; experimental research; experimental study; gene product; genetic regulatory protein; in vivo; language translation; living system; mutant; paralog; paralogous gene; protein B; regulatory gene product; research study; senescence; senescent; social role; telomerase RNA; telomerase catalytic subunit; telomerase reverse transcriptase; telomerase reverse transcriptase catalytic subunit; telomere; tool; tumor; yeast two hybrid system
Project start date: 1993-09-20
Project end date: 2013-01-31
Budget start date: 15-FEB-2010
Budget end date: 31-JAN-2011
5R37AG011728-17 (2010): $388649
5R37AG011728-14 (2007): $404921
5R37AG011728-13 (2006): $417015
5R37AG011728-11 (2004): $338625
Grants awarded to Victoria Lundblad
YEAST CHROMOSOME STRUCTURE, REPLICATION AND SEGREGATION
Victoria Lundblad, Associate Professor
Federation Of Amer Soc For Exper Biology
9650 Rockville Pike
bethesda, Md 208143998
Grant 1R13CA087833-01 from National Cancer Institute IRG: ZCA1
Abstract: This proposal requests partial support for a Federation of American Societies of Experimental Biology (FASEB) summer research conference on Yeast Chromosome Structure, Replication and Segregation. This meeting will be held August 19-24, 2000 at Snowmass, Colorado, as the sixth meeting in an extremely successful biennial series on chromosome research in budding and fission yeast. Research on yeast chromosome dynamics has a substantial impact on human biology, with medical implications in the areas of cancer, cellular aging, and aneuploidy due to chromosome transmission defects. Therefore, this rapidly moving research area requires a dedicated meeting to efficiently disseminate information. Since 1990, the FASEB-sponsored meeting has been the most important clearinghouse for new experimental findings in this field. The 2000 conference program will consist of nine scientific sessions, a keynote address, a workshop, and two poster sessions. The scientific sessions will cover nuclear architecture and chromatin remodeling, DNA replication, cell cycle control and checkpoints, telomeres and position effects, centromeres and their interacting proteins, components of the mitotic spindle apparatus, microtubule motors, meiotic chromosome segregation and chromosome cohesion. The focus of the afternoon- long workshop will be yeast functional genomics, a topic of increasing interest due to the tremendous potential impact on genetic analysis. Ten distinguished scientists have been invited to participate in this conference as session chairs of the nine plenary sessions and the workshop. Dr. Kim Nasmyth (I.M.P., Vienna, Austria), a leader in the field o sister chromatid separation, has been invited to give the keynote address. In consultation with the session chairs, we have generated a list of 104 potential speakers from which we will make a final selection of approximately 66. Most speakers will be invited in Sept./Oct. 1999, while we plan to choose others from among the submitted s prior to the meeting. Participants whose s were not selected for platform talks will be strongly encouraged to present posters. The conference will be limited to 200 scientists who will be chosen on the basis of their interests and expertise, with special efforts to increase the participation of female, minority and junior scientists. Our objective for this meeting is to continue the extremely high level of excitment and scientific excellence that has characterized the past five conferences
Keywords: DNA replication, chromosome, chromosome movement, fungal genetics, meeting /conference /symposium, nucleic acid structure, yeast travel
Project start date: 2000-05-01
Project end date: 2001-04-30
1R13CA087833-01 (2000): $5000
DUAL ROLE FOR CDC13 IN YEAST TELOMERE FUNCTION
Victoria Lundblad, Associate Professor
Baylor College Of Medicine 1 Baylor Plaza Houston, Tx 770303498
Grant 1R01GM055867-01 from National Institute Of General Medical Sciences IRG: CTY
Abstract: This application focusses on CDC13, a gene that had previously been shown by Hartwell s laboratory to have an essential role in maintaining telomere integrity. The PI has since shown that Cdc13 is a single-strand binding protein, suggesting that the essential function of this protein is to provide a protective cap at the end of the chromosome. However, the PI has also shown that CDC13 has a role in the telomerase-mediated pathway for telomere maintenance, leading her to propose that the Cdc13 protein regulates telomerase by mediating access of this enzyme to the chromosome terminus. This proposal sets out to examine these two roles for the Cdc13 protein by a combination of genetic and biochemical techniques. The specific aims are (1) to use three different genetic screens to look for factors that interact with CDC13 either directly or indirectly. (2) To conduct a detailed functional analysis of CDC13 via analysis of the DNA-binding domain, analysis of the telomerase-related function, and identification of conserved and/or hypomutable regions of the gene. (3) To conduct a biochemical analysis of the proposed role of the Cdc13 protein by examining whether Cdc13 and associated proteins are components of telomerase or telomeric chromatin, by characterizing the interactions between Cdc13 and the products of genes identified in aim 1, and by characterizing any enzymatic activities associated with either Cdc13 or Cdc13 interacting proteins.
Keywords: DNA binding protein, fungal genetics, protein structure /function, telomere, chromatin, enzyme activity, telomerase, nucleic acid sequence, polymerase chain reaction, tissue /cell culture
Project start date: 1997-05-01
Project end date: 2001-04-30
1R01GM055867-01 (1997): $204528
5R01GM055867-04 (2000): $238589
5R01GM055867-03 (1999): $231725
TELOMERE REPLICATION AND SENESCENCE IN YEAST
Victoria Lundblad, Associate Professor
Molecular And Human Geneticsbaylor College Of Medicine
1 Baylor Plaza
houston, Tx 770303498
Grant 5R01AG011728-09 from National Institute On Aging IRG: CTY
Abstract: The Lundblad lab is using a genetic approach in the budding yeast Saccharomyces cerevisiae to identify potential components or positive regulators of telomerase. During the previous grant period the lab conducted a genetic screen for yeast mutants that displayed a similar phenotype to the previously identified est1- mutant. This mutant displays progressive telomere shortening and a senescence phenotype. The genetic screen resulted in identification of two new EST genes (EST2 and EST3) as well as a novel est-like mutation in a gene previously implicated at the telomere (CDC13). The CDC13 and EST gene products are essential in vivo for telomere replication but are dispensable in vitro for telomerase activity. Thus, they may be essential regulators of telomerase rather than part of the core enzyme. Since Est1p and Cdc13p are both single-strand telomere binding proteins, the PI proposes that these two proteins function to mediate access of telomerase to the chromosomal terminus. Est2p and Est3p could act similarly as components of telomeric chromatin, or as other in vivo regulators of telomerase. This application proposes to take two inter-related approaches to further analysis of the EST/TLC1 pathway for telomere replication. The first is to extensively characterize each EST gene with the long term goal of determining what each individual protein is doing at the telomere and using this information to ultimately reconstitute telomerase activity in vivo. Specifically, the lab will analyze the biochemical properties of the Est1 protein; look for potential interactions between the individual Est proteins, as well as between the Est proteins and other gene products; determine whether the Est proteins form a complex and are components of either telomeric chromatin or telomerase; reconstitute yeast telomerase activity. In a parallel approach, the lab will conduct several extensive mutant screens designed to identify additional genes required for telomere function in yeast including additional EST genes. In this aim the lab will perform a new mutant screen designed to identify additional genes in the EST/TLC pathway for telomere replication; screen the 4900 viable yeast null mutations for changes in telomere length; characterize and clone the genes mutated in a new collection of 25 short and long telomere mutants. The genes identified in these screens will be incorporated into the ongoing genetic and biochemical experiments with the existing EST genes
Keywords: DNA replication, cell senescence, enzyme activity, fungal genetics, genetic regulation, telomerase, telomere DNA binding protein, Saccharomyces cerevisiae, gene mutation, nucleic acid sequence, protein structure function molecular cloning, yeast two hybrid system
Project start date: 1993-09-20
Project end date: 2002-12-14
5R01AG011728-09 (2001): $259303
2R01AG011728-05 (1997): $244114
5R01AG011728-07 (1999): $244422
2R37AG011728-10 (2003): $338625
Dual Role For CDC13 In Yeast Telomere Function
Victoria Lundblad, Associate Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 7R01GM055867-09 from National Institute Of General Medical Sciences IRG: CDF
Abstract: Telomeres, the structures found at the ends of linear chromosomes, are essential for genomic stability and normal cellular growth. The past last few years has seen an explosive interest in the possibility that telomere length regulation is involved in the related processes of aging and cancer. Therefore, a complete understanding of the components that are required for normal telomere function is necessary, in order to address the consequences of telomere dysfunction in abnormal cells. Telomere function in both yeast and human cells requires factors that are essential for maintaining the integrity of the chromosome terminus, since loss ol chromosome end protection is immediately lethal for the cell. In addition, the enzyme telomerase, which is responsible for replicating the ends of chromosomes, is necessary for long term cellular proliferation. In yeast, the single-strand telomere binding protein Cdc 13 is critical for both telomere end protection and telomere replication certain mutations in CDC13 result in extensive degradation of the end of the chromosome, whereas other mutations in Cdcl3 prevent telomerase access to the telomere. This application proposes an inter-related set of experiments designed to analyze the dual role of CDCI 3 in these two processes. Both genetic and biochemical experiments will be used to identify other proteins that function in combination with Cdcl3 to protect the telomere; such experiments also have the potential to identify the activity(s) that are responsible for degrading unprotected telomeres. In parallel, a set of experiments is presented to analyze how telomerase is recruited to the chromosome by Cdcl3, with a focus on the behavior of two proteins that mediate how Cdcl3 regulates telomerase access to the telomere. Characterization of the dual nature of this essential yeast telomere protein may serve as a paradigm for similar events at mammalian telomeres.
Keywords: DNA binding protein, fungal genetics, protein structure function, telomere, chromatin, enzyme activity, telomerase
Project start date: 1997-05-01
Project end date: 2007-05-31
2R01GM055867-05 (2001): $273910
Sponsored Links Excellgen http://Excellgen.com
5R01GM055867-08 (2004): $273910
5R01GM055867-07 (2003): $273910
DNA END-JOINING PROTEINS AND TELOMERE FUNCTION
Victoria Lundblad, Associate Professor
Molecular And Human Geneticsbaylor College Of Medicine
1 Baylor Plaza
houston, Tx 770303498
Grant 3R01AG016626-03S1 from National Institute On Aging IRG: ZAG1
Abstract: Telomeres, the ends of eukaryotic chromosomes, are essential structures that mediate complete replication of chromosomal termini and cellular proliferation, and also insulate chromosome ends from degradation and end-to-end fusions. Studies in both yeast and mammalian cells have shown that the inability to replicate the telomere leads to telomere shortening and cellular senescence. Therefore, genes that ensure normal telomere function fall into the category of longevity assurance genes. The primary activity responsible for telomere replication is the enzyme telomerase; loss of this enzyme results in telomere shortening and an inability to proliferate. In contrast, the exact molecular mechanism by which protection of telomeres occurs is unclear. We have recently demonstrated that proteins that have been well characterized for their requirement in double strand break repair also play critical roles in telomere function. We have proposed that one of these protein complexes, the Ku heterodimer, is required to protect chromosomal termini from degradation; this activity is required in parallel with telomerase, as strains of yeast defective for both telomerase and Ku exhibit a greatly accelerated senescence phenotype. The second DNA repair complex, the Mre11/Rad5O/Xrs2 complex, plays a different role at the telomere, in that it is required for the telomerase-mediated pathway for telomere replication, rather than for end protection. These results pose a fundamental question regarding how recruitment of Ku and Mre11/Rad50/Xrs2 to both random double strand breaks and the telomere can have such different consequences in vivo if the same proteins that mediate repair of DNA strand breaks are also present at the telomere, what prevents telomeres from similarly undergoing end-to-end fusions? We hypothesize that these repair proteins have activity(s) at the telomere that are distinct from their action at double strand breaks. We propose to address this hypothesis via the identification of mutants in these DNA repair genes that uncouple telomere function from double- strand break repair. Second, we present molecular and biochemical assays designed to characterize the roles of Ku and Mre11/Rad50/Xrs2 at the telomere. Finally, we propose to use several specific genetic screens to identify and characterize new genes that function with Ku and the Mre11/Rad5O/Xrs2 complex at the telomere
Keywords: DNA binding protein, DNA repair, gene expression, protein structure function, telomere DNA damage, DNA replication, enzyme activity, gene mutation, phenotype, protein binding, telomerase polymerase chain reaction, yeast two hybrid system
Project start date: 1999-04-01
Project end date: 2004-03-31
3R01AG016626-03S1 (2001): $130118
5R01AG016626-03 (2001): $289278
5R01AG016626-02 (2000): $280146
1R01AG016626-01 (1999): $299152
5R01AG016626-05 (2003): $306894
TELOMERE REPLICATION AND SENESCENCE IN YEAST
Victoria Lundblad, Associate Professor
Baylor College Of Medicine
1 Baylor Plaza
houston, Tx 770303498
Grant 1R01AG011728-01 from National Institute On Aging IRG: CTY
Abstract: Telomeres are specialized elements found at the termini of linear chromosomes. They are required to protect chromosome ends from degradation and fusion with other ends; in addition, telomeres also promote accurate chromosome segregation. In humans, missegregation of chromosomes can lead to a number of disorders, such as cancer and birth defects. Defects in telomere replication in yeast and Tetrahymena lead to senescence, suggesting that this could be a contributing factor in mammalian cellular senescence as well. A primary determinant for maintaining a constant chromosomal telomere length is telomerase, identified biochemically in ciliates and humans. Telomerase has been shown to be a novel reverse transcriptase-like enzyme which carries an internal RNA responsible for templating the newly synthesized telomeric DNA. Telomerase activity has not yet been biochemically identified from a genetically tractable organism such as yeast, which has limited the analysis of factors that interact with this enzyme. In addition, nothing is yet known about the protein composition of telomerase. The long term objective of the proposed research is to dissect the apparatus responsible for replicating telomeres, using the yeast S. cerevisiae as a model system. A starting point for these studies is the EST1 gene of yeast. Yeast cells lacking a functional EST1 gene show a continuous decline in telomere length and a senescence phenotype. Although a number of mutants of yeast have been identified which alter telomere length, this is the only mutant of yeast which coordinately affects both telomere length and senescence. No exhaustive search has previously been done in any organism for genes which, when mutated, coordinately affect telomere replication and senescence. S. cerevisiae lends itself to such a search, both because it is a genetically tractable system and because isolation of the est1 mutation provides a precedent for this particular class of mutations. The first section of this proposal describes a genetic approach to identify all other genes besides EST1 which, when mutated, coordinately affect telomere replication and senescence; this should include the predicted yeast telomerase RNA Recovery of such genes is an important step towards understanding how different components involved in telomere replication contribute to cellular senescence in a lower eukaryote. The second aim of this research is to identify additional genes that encode components that interact with the EST1 gene product, using three different genetic approaches; characterization of these genes should provide additional information about Est1 function. A third goal is to biochemically characterize activities associated with the Est1 protein. Two hypotheses will guide these biochemical experiments first, that Est1 is a component of telomerase, and second, that Est1 is a telomere binding protein
Keywords: DNA replication, cell senescence, fungal genetics, reverse transcriptase, telomere DNA binding protein, Saccharomyces cerevisiae, gene mutation, nucleic acid sequence genetic manipulation
Project start date: 1993-09-20
Project end date: 1997-08-31
1R01AG011728-01 (1993): $177813
CAUSATIVE GENETIC VARIATION IN HUMAN TELOMERE GENES
Victoria Lundblad, Associate Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 5R01AG024402-04 from National Institute On Aging IRG: ZAG1
Abstract: Telomere length can vary substantially among individuals, and recent data show that this length variation correlates with age-dependent mortality from infectious and cardiovacular diseases. Since in humans, as in all eukaryotic organisms with linear chromosomes, telomere length depends on a highly conserved mechanism for maintenance of chromosome termini, our ultimate goal is to test whether natural genetic variation in multiple telomere maintenance genes contributes to age-related diseases and mortality. However, identifying such causative variation in multiple genes, by comparative standard SNP analysis of age-matched cohorts, is a formidable challenge. This application targets a subset of telomere maintenance genes that are conserved in yeast and humans, and presents a potential methodological solution to the problem of identifying causative genetic variation. A multidisciplinary approach combines a new computational method, called the Evolutionary Trace (ET), that identifies patterns of sequence variation that correlate with functional divergence, with in vivo modeling in yeast cells of the "fitness" of genetic variation in genes that are required for telomere function. Our goals are to test whether a functional correlation can be established between naturally occurring genetic variation in conserved telomere maintenance genes and effects on cellular proliferation; and whether ET can identify functionally important cSNPs in the human versions of the 8 telomere maintenance genes selected for this study. If so, this will enable larger scale telomere maintenance/disease-association studies that will now be feasible because they will focus on a subset of polymorphisms. More generally, it will provide proof of principle that human polymorphisms can be initially screened computationally for their functional importance based on evolutionary analysis. This will, we hope, set the stage for elucidating the target(s) of telomere-based human aging, with the potential for clinical intervention to alleviate the onset of at least some age-dependent diseases.
Keywords: aging, computational biology, disease /disorder onset, functional /structural genomics, genetic regulation, longevity, method development, single nucleotide polymorphism, telomere, biochemical evolution, cell proliferation, cytogenetics, fungal genetics, gene expression, genetic model, intermolecular interaction, nucleic acid structure, protein structure function, Saccharomyces cerevisiae, green fluorescent protein, human genetic material tag, phenotype, polymerase chain reaction
Project start date: 2004-08-15
Project end date: 2007-07-31
5R01AG024402-04 (2006): $298073
5R01AG024402-03 (2005): $296352
Sponsored Links Excellgen http://Excellgen.com
1R01AG024402-01 (2004): $309770
Telomere Replication And Senescence In Yeast
Victoria Lundblad, Associate Professor
Salk Institute For Biological Studies La Jolla, Ca 920371099
Grant 7R37AG011728-12 from National Institute On Aging IRG: CDF
Project start date: 1993-09-20
Project end date: 2007-11-30
7R37AG011728-12 (2005): $427050
TRAINING PROGRAM IN CELL AND MOLECULAR BIOLOGY
Victoria Lundblad, Associate Professor
Baylor College Of Medicine 1 Baylor Plaza Houston, Tx 770303498
Grant 5T32GM008231-14 from National Institute Of General Medical Sciences IRG: BRT
Project start date: 1990-07-01
Project end date: 2005-06-30
5T32GM008231-14 (2003): $290084
5T32GM008231-13 (2002): $270515
5T32GM008231-12 (2001): $250470
2T32GM008231-11 (2000): $234918
TELOMERE REPLICATION AND SENESCENCE IN YEAST
Victoria Lundblad, Associate Professor
Inst For Molecular Geneticsbaylor College Of Medicine
1 Baylor Plaza
houston, Tx 770303498
Grant 5R01AG011728-04 from National Institute On Aging IRG: CTY
Project start date: 1993-09-20
Project end date: 1997-08-31
5R01AG011728-04 (1996): $182727