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Biomedical subjects

V Lundblad

Publications and source records attributed to V Lundblad.

At least 19 recordsLinked to original sources
PubMed2000

DNA ends: maintenance of chromosome termini versus repair of double strand breaks.

This review focuses on the factors that define the differences between the two types of DNA ends encountered by eukaryotic cells: telomeres and double strand breaks (DSBs). Although these two types of DNA termini are functionally distinct, recent studies have shown that a number of proteins is shared at telomeres and sites of DSB repair. The significance of these common components is discussed, as well as the types of DNA repair events that can compensate for a defective telomere.

Chromosomes↗
PubMed2000

The Est3 protein is a subunit of yeast telomerase.

EST1, EST2, EST3 and TLC1 function in a single pathway for telomere replication in the yeast Saccharomyces cerevisiae [1] [2], as would be expected if these genes all encode components of the same complex. Est2p, the reverse transcriptase protein subunit, and TLC1, the templating RNA, are subunits of the catalytic core of yeast telomerase [3] [4] [5]. In contrast, mutations in EST1, EST3 or CDC13 eliminate telomere replication in vivo [1] [6] [7] [8] but are dispensable for in vitro telomerase catalytic activity [2] [9]. Est1p and Cdc13p, as components of telomerase and telomeric chromatin, respectively, cooperate to recruit telomerase to the end of the chromosome [7] [10]. However, Est3p has not yet been biochemically characterized and thus its specific role in telomere replication is unclear. We show here that Est3p is a stable component of the telomerase holoenzyme and furthermore, association of Est3p with the enzyme requires an intact catalytic core. As predicted for a telomerase subunit, fusion of Est3p to the high affinity Cdc13p telomeric DNA binding domain greatly increases access of telomerase to the telomere. Est1p is also tightly associated with telomerase; however, Est1p is capable of forming a stable TLC1-containing complex even in the absence of Est2p or Est3p. Yeast telomerase therefore contains a minimum of three Est proteins for which there is both in vivo and in vitro evidence for their role in telomere replication as subunits of the telomerase complex.

Binding Sites↗
PubMed2000

Molecular biology. Telomeres keep on rappin'.

Many molecules help maintain the ends of chromosomes, which get chewed off as cells age. Lundblad in her provocative Perspective now tells us about another protein, hRap1, that regulates the length of telomeres in human cells with the help of the TRF proteins. The homology between hRap1 and its counterpart in yeast suggests how the complex molecular machinery needed to maintain chromosome ends may have evolved.

Caenorhabditis elegans Proteins↗
PubMed2000

Identification of the single-strand telomeric DNA binding domain of the Saccharomyces cerevisiae Cdc13 protein.

The CDC13 gene of Saccharomyces cerevisiae is required both to protect telomeric DNA and to ensure proper function of yeast telomerase in vivo. We have previously demonstrated that Cdc13p has a high affinity single-strand telomeric DNA binding activity, although the primary amino acid sequence of Cdc13p has no previously characterized DNA binding motifs. We report here mapping of the Cdc13 DNA binding domain by a combination of proteolysis mapping and deletion cloning. The DNA binding domain maps to residues 557-694 of the 924-amino acid Cdc13 polypeptide, within the most basic region of Cdc13p. A slightly larger version of this domain can be efficiently expressed in Escherichia coli as a soluble small protein, with DNA binding properties comparable to those of the full-length protein. A single amino acid missense mutation within this domain results in thermolabile DNA binding and conditional lethality in yeast, consistent with the prediction that DNA binding should be essential for CDC13 function. These results show that Cdc13p contains a discrete substructure responsible for DNA binding and should facilitate structural characterization of this telomere binding protein.

Binding Sites↗
PubMed2000

Positive and negative regulation of telomerase access to the telomere.

The protective caps on chromosome ends - known as telomeres - consist of DNA and associated proteins that are essential for chromosome integrity. A fundamental part of ensuring proper telomere function is maintaining adequate length of the telomeric DNA tract. Telomeric repeat sequences are synthesized by the telomerase reverse transcriptase, and, as such, telomerase is a central player in the maintenance of steady-state telomere length. Evidence from both yeast and mammals suggests that telomere-associated proteins positively or negatively control access of telomerase to the chromosome terminus. In yeast, positive regulation of telomerase access appears to be achieved through recruitment of the enzyme by the end-binding protein Cdc13p. In contrast, duplex-DNA-binding proteins assembled along the telomeric tract exert a feedback system that negatively modulates telomere length by limiting the action of telomerase. In mammalian cells, and perhaps also in yeast, binding of these proteins probably promotes a higher-order structure that renders the telomere inaccessible to the telomerase enzyme.

Animals↗
PubMed1999

Est1 and Cdc13 as comediators of telomerase access.

Cdc13 and Est1 are single-strand telomeric DNA binding proteins that contribute to telomere replication in the yeast Saccharomyces cerevisiae. Here it is shown that fusion of Cdc13 to the telomerase-associated Est1 protein results in greatly elongated telomeres. Fusion proteins consisting of mutant versions of Cdc13 or Est1 confer similar telomere elongation, indicating that close physical proximity can bypass telomerase-defective mutations in either protein. Fusing Cdc13 directly to the catalytic core of telomerase allows stable telomere maintenance in the absence of Est1, consistent with a role for Est1 in mediating telomerase access. Telomere length homeostasis therefore is maintained in part by restricting access of telomerase to chromosome termini, but this limiting situation can be overcome by directly tethering telomerase to the telomere.

Binding Sites↗
PubMed1999

Assembly and regulation of telomerase.

Recent studies on the telomerase reverse transcriptase have benefited from the identification of the catalytic core subunits. Cellular factors that participate in the assembly of the core enzyme have been identified and regulatory mechanisms that control telomerase activity are beginning to be elucidated.

Animals↗
PubMed1998

Telomere maintenance is dependent on activities required for end repair of double-strand breaks.

Telomeres are functionally distinct from ends generated by chromosome breakage, in that telomeres, unlike double-strand breaks, are insulated from recombination with other chromosomal termini [1]. We report that the Ku heterodimer and the Rad50/Mre11/Xrs2 complex, both of which are required for repair of double-strand breaks [2-5], have separate roles in normal telomere maintenance in yeast. Using epistasis analysis, we show that the Ku end-binding complex defined a third telomere-associated activity, required in parallel with telomerase [6] and Cdc13, a protein binding the single-strand portion of telomere DNA [7,8]. Furthermore, loss of Ku function altered the expression of telomere-located genes, indicative of a disruption of telomeric chromatin. These data suggest that the Ku complex and the Cdc13 protein function as terminus-binding factors, contributing distinct roles in chromosome end protection. In contrast, MRE11 and RAD50 were required for the telomerase-mediated pathway, rather than for telomeric end protection; we propose that this complex functions to prepare DNA ends for telomerase to replicate. These results suggest that as a part of normal telomere maintenance, telomeres are identified as double-strand breaks, with additional mechanisms required to prevent telomere recombination. Ku, Cdc13 and telomerase define three epistasis groups required in parallel for telomere maintenance.

Antigens, Nuclear↗
PubMed1998

Telomerase, Ku, and telomeric silencing in Saccharomyces cerevisiae.

Telomeres comprise a specialized chromosome end structure distinct from the standard nucleosomal architecture of the remainder of the genome. Telomere maintenance and chromosome stability require both replication of telomeric sequences by telomerase and telomeric end protection through binding of proteins. We have shown that Cdc13p and the heterodimer Ku are required, along with telomerase, for full telomere function, and we have proposed that Ku and Cdc13p contribute distinct roles in end protection. Ku has recently been shown to exhibit defects in transcriptional repression of telomere-proximal genes, known as telomere position effect (TPE), or telomeric silencing. We investigate here whether alterations in genes involved in the telomerase pathway also exhibit TPE defects and find that deletion or overexpression of EST1 or EST2 does not significantly affect telomeric silencing. However, telomeric silencing is derepressed upon overexpression of certain nonfunctional alleles of each. In addition, we determined that overproduction of telomerase pathway components partially alleviates the TPE defect in hdf1Delta cells. This indicates that there is genetic crosstalk between these two telomere maintenance pathways, and suggests that overproduction of telomerase pathway components may at least partially compensate for the loss of Ku in maintaining telomeric silencing.

Antigens, Nuclear↗
PubMed1998

Telomeres and double-strand breaks: trying to make ends meet.

Eukaryotic cells encounter two types of DNA ends: telomeres, the natural ends of linear chromosomes, and double-strand breaks, resulting from DNA damage or normal chromosomal processes such as meiotic or V(D)J recombination. These two termini have long been seen as functionally distinct, based on whether they are resistant to fusion with other ends or instead are acted upon by the DNA-repair machinery. However, a series of recent papers has shown that members of a set of proteins that are crucial for the rejoining of DNA strand breaks are also required for normal telomere function, raising new questions about how these two types of termini maintain their functional distinction.

Antigens, Nuclear↗
PubMed1997

Programmed translational frameshifting in a gene required for yeast telomere replication.

BACKGROUND: Telomeres are replicated in most eukaryotes by the enzyme telomerase, a specialized reverse transcriptase. A genetic screen in Saccharomyces cerevisiae designed to detect telomerase components previously led to the identification of four EST ('ever shorter telomeres') genes which are required for telomerase function in vivo. This report describes the cloning and characterization of EST3. RESULTS: We identified a potential site of +1 ribosomal frameshifting in the EST3 coding sequence and demonstrated that translation both upstream and downstream of this site is required for EST3 function. Mutation of EST3 such that it could not frameshift resulted in a strain with the same phenotype as an est3 null mutant, showing that EST3 frameshifting is required for telomere replication. Immunoblot analysis revealed that two proteins were synthesized from EST3: a truncated protein resulting from translation of only the first open reading frame, as well as the full-length 181 amino-acid Est3 protein resulting from translation through the frameshift site. Only the full-length Est3 protein was required for normal EST3 function. CONCLUSIONS: A programmed translational frameshifting mechanism similar to that used by yeast retrotransposons is employed to produce full-length Est3 protein. This is the first example in yeast of a cellular gene that uses frameshifting to make its protein product, and a potential link is suggested between retrotransposition and the telomerase pathway for telomere maintenance.

Amino Acid Sequence↗
PubMed1997

Three Ever Shorter Telomere (EST) genes are dispensable for in vitro yeast telomerase activity.

Telomerase is a specialized reverse transcriptase consisting of both RNA and protein components. Previous characterization of yeast telomerase function in vivo identified four EST (for ever shorter telomeres) genes that, when mutated, result in the phenotypes expected for a defect in telomerase. Consistent with this genetic prediction, the EST2 gene has recently been shown to encode the catalytic component of telomerase. Using an in vitro assay, we show here that telomerase activity is present in extracts prepared from yeast strains carrying est1-Delta, est3-Delta, and cdc13-2(est) mutations. Therefore, while these three genes are necessary for telomerase function in vivo, they do not encode components essential for core catalytic activity. When Est2p, the one EST gene product found to be essential for catalytic activity, was immunoprecipitated from extracts, the telomerase RNA subunit was also specifically precipitated, supporting the conclusion that these two components are in a stable complex.

Centrifugation, Density Gradient↗