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Victoria Lundblad

Publications and source records attributed to Victoria Lundblad.

11 recordsLinked to original sources
PubMed2004

Structural elements required for association of the Saccharomyces cerevisiae telomerase RNA with the Est2 reverse transcriptase.

Telomere synthesis in most organisms depends on the action of the telomerase enzyme, which contains an RNA subunit that is stably associated with the reverse transcriptase subunit as well as additional telomerase proteins. In the budding yeast Saccharomyces cerevisiae, several structural domains that are responsible for mediating protein interactions with the telomerase RNA TLC1 have been identified. We report here the identification and characterization of a TLC1 stem-loop that is required for its interaction with the Est2 reverse transcriptase protein. This hairpin, which does not contain any bulges in the duplex stem that commonly mediate protein-RNA interaction, appears to be a part of a larger structure, as nucleotides immediately to either side of this stem-loop contribute to the interaction of TLC1 with the Est2 protein. Surprisingly, replacement of a 95-nucleotide region of the yeast telomerase RNA that is required for Est2 interaction with a 39-nucleotide pseudoknot from a distantly related telomerase RNA results in a functional telomerase enzyme. These findings suggest that the ability of the budding yeast reverse transcriptase to associate with the telomerase RNA depends on a highly structured region rather than specific sequence elements.

Animals↗
PubMed2004

EXO1 contributes to telomere maintenance in both telomerase-proficient and telomerase-deficient Saccharomyces cerevisiae.

Previous work in budding yeast has indicated that telomeres are protected, at least in part, from the action of Exo1, which degrades the C-rich strand of partially uncapped telomeres. To explore this further, we examined the consequences of Exo1-mediated activity in strains that lacked Ku, telomerase, or both. Loss of Exo1 partially rescued the telomere length defect in a yku80delta strain, demonstrating that exonuclease action can directly contribute to telomere shortening. The rapid loss of inviability displayed by a yku80delta est2delta strain was also partially alleviated by an exo1delta mutation, further supporting the proposal that Exo1 is one target of the activities that normally protect wild-type telomeres. Conversely, however, Exo1 activity was also capable of enhancing telomere function and consequently cell proliferation, by contributing to a telomerase-independent pathway for telomere maintenance. The recovery of recombination-dependent survivors that arose in a yku80delta est2delta strain was partially dependent on Exo1 activity. Furthermore, the types of recombination events that facilitate telomerase-independent survival were influenced by Exo1 activity, in both est2delta and yku80delta est2delta strains. These data demonstrate that Exo1 can make either positive or negative contributions to telomere function and cell viability, depending on whether telomerase or recombination is utilized to maintain telomere function.

Cell Proliferation↗
PubMed2003

Homology among telomeric end-protection proteins.

Telomere maintenance and end protection are essential for the survival and proliferation of eukaryotic cells, leading to the prediction that components of this system would be highly conserved. In practice, however, evidence for homology among these factors has been elusive, and, in the case of the known end-protection proteins, evolutionary relationships have been postulated largely on the basis of protein structural and functional similarity alone. Here we report support from sequence profile analyses for a significant and specific evolutionary relationship among OB-fold telomeric end-protection factors.

Databases, Protein↗
PubMed2003

Telomerase-independent proliferation is influenced by cell type in Saccharomyces cerevisiae.

Yeast strains harboring mutations in genes required for telomerase function (TLC1 and the EST genes) exhibit progressive shortening of telomeric DNA and replicative senescence. A minority of cells withstands loss of telomerase through RAD52-dependent amplification of telomeric and subtelomeric sequences; such survivors are now capable of long-term propagation with telomeres maintained by recombination rather than by telomerase. Here we report that simultaneous expression in haploid cells of both MATa and MATalpha information suppresses the senescence of telomerase-deficient mutants, with suppression occurring via the RAD52-dependent survivor pathway(s). Such suppression can be mimicked by deletion of SIR1-SIR4, genes that function in transcriptional silencing of several loci including the silent mating-type loci. Furthermore, telomerase-defective diploid strains that express only MATa or MATalpha information senesce at a faster rate than telomerase-defective diploids that are heterozygous at the MAT locus. This suggests that the RAD52-dependent pathway(s) for telomere maintenance respond to changes in the levels of recombination, a process regulated in part by the hierarchy of gene control that includes MAT regulation. We propose that cell-type-specific regulation of recombination at human telomeres may similarly contribute to the tissue-specific patterns of disease found in telomerase-deficient tumors.

Aging↗
PubMed2003

The Ku heterodimer performs separable activities at double-strand breaks and chromosome termini.

The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80(tel) alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80(tel) alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

Alleles↗
PubMed2002

Conserved structure for single-stranded telomeric DNA recognition.

The essential Cdc13 protein in the yeast Saccharomyces cerevisiae is a single-stranded telomeric DNA binding protein required for chromosome end protection and telomere replication. Here we report the solution structure of the Cdc13 DNA binding domain in complex with telomeric DNA. The structure reveals the use of a single OB (oligonucleotide/oligosaccharide binding) fold augmented by an unusually large loop for DNA recognition. This OB fold is structurally similar to OB folds found in the ciliated protozoan telomere end-binding protein, although no sequence similarity is apparent between them. The common usage of an OB fold for telomeric DNA interaction demonstrates conservation of end-protection mechanisms among eukaryotes.

Binding Sites↗
PubMed2002

Telomere maintenance without telomerase.

Recombination-dependent maintenance of telomeres, first discovered in budding yeast, has revealed an alternative pathway for telomere maintenance that does not require the enzyme telomerase. Experiments conducted in two budding yeasts, S. cerevisiae and K. lactis, have shown recombination can replenish terminal G-rich telomeric tracts that would otherwise shorten in the absence of telomerase, as well as disperse and amplify sub-telomeric repeat elements. Investigation of the genetic requirements for this process have revealed that at least two different recombination pathways, defined by RAD50 and RAD51, can promote telomere maintenance. Although critically short telomeres are very recombinogenic, recombination among telomeres that have only partially shortened in the absence of telomerase can also contribute to telomerase-independent survival. These observations provide new insights into the mechanism(s) by which recombination can restore telomere function in yeast, and suggest future experiments for the investigation of potentially similar pathways in human cells.

DNA Replication↗
PubMed2002

Mechanisms of chromosome-end protection.

Telomeres must protect chromosome ends from being recognized and processed as double-strand breaks. Identification of the factors involved in end protection, and the mechanisms by which they "cap" chromosome termini, is crucial in understanding how the cell distinguishes between a double-strand break and a normal telomere end. Recent work has characterized the similarities and potential differences between the pathways utilized by multiple organisms in maintaining telomere ends. One unifying concept that has clearly emerged is that chromosome-end protection is necessary in maintaining genetic stability and preventing oncogenesis.

Antigens, Nuclear↗
PubMed2002

The Est1 subunit of Saccharomyces cerevisiae telomerase makes multiple contributions to telomere length maintenance.

The telomerase-associated Est1 protein of Saccharomyces cerevisiae mediates enzyme access by bridging the interaction between the catalytic core of telomerase and the telomere-binding protein Cdc13. In addition to recruiting telomerase, Est1 may act as a positive regulator of telomerase once the enzyme has been brought to the telomere, as previously suggested by the inability of a Cdc13-Est2 fusion protein to promote extensive telomere elongation in an est1-Delta strain. We report here three classes of mutant Est1 proteins that retain association with the telomerase enzyme but confer different in vivo consequences. Class 1 mutants display a telomere replication defect but are capable of promoting extensive telomere elongation in the presence of a Cdc13-Est2 fusion protein, consistent with a defect in telomerase recruitment. Class 2 mutants fail to elongate telomeres even in the presence of the Cdc13-Est2 fusion, which is the phenotype predicted for a defect in the proposed second regulatory function of EST1. A third class of mutants impairs an activity of Est1 that is potentially required for the Ku-mediated pathway of telomere length maintenance. The isolation of mutations that perturb separate functions of Est1 demonstrates that a telomerase holoenzyme subunit can contribute multiple regulatory roles to telomere length maintenance.

Amino Acid Sequence↗