Wiley, EA, and VA Zakian. “
Extra telomeres, but not internal tracts of telomeric DNA, reduce transcriptional repression at Saccharomyces telomeres.”.
Genetics 139.1 (1995): ,
139, 1, 67-79. Print.
AbstractYeast telomeric DNA is assembled into a nonnucleosomal chromatin structure known as the telosome, which is thought to influence the transcriptional repression of genes placed in its vicinity, a phenomenon called telomere position effect (TPE). The product of the RAP1 gene, Rap1p, is a component of the telosome. We show that the fraction of cells exhibiting TPE can be substantially reduced by expressing large amounts of a deletion derivative of Rap1p that is unable to bind DNA, called Rap1 delta BBp, or by introducing extra telomeres on a linear plasmid, presumably because both compete in trans with telomeric chromatin for factor(s) important for TPE. This reduction in TPE, observed in three different strains, was demonstrated for two different genes, each assayed at a different telomere. In contrast, the addition of internal tracts of telomeric DNA on a circular plasmid had very little effect on TPE. The product of the SIR3 gene, Sir3p, appears to be limiting for TPE. Overexpression of Sir3p completely suppressed the reduction in TPE observed with expression of Rap1 delta BBp, but did not restore high levels of TPE to cells with extra telomeres. These results suggest that extra telomeres must titrate a factor other than Sir3p that is important for TPE. These results also provide evidence for a terminus-specific binding factor that is a factor with a higher affinity for DNA termini than for nonterminal tracts of telomeric DNA and indicate that this factor is important for TPE.
Lin, JJ, and VA Zakian. “
An in vitro assay for Saccharomyces telomerase requires EST1.”.
Cell 81.7 (1995): ,
81, 7, 1127-35. Print.
AbstractTelomerase activity was demonstrated in cell-free extracts from S. cerevisiae through the use of a PCR-based assay. As expected, this activity was eliminated by RNase or phenol treatment of the extract and was dependent on dGTP and dTTP. Telomerase was not detected in extracts prepared from cells grown for approximately 30 or more cell divisions in the absence of the EST1 product, Est1p. TLC1 RNA, which determines the sequence of telomeric DNA in vivo, was present in normal amounts in est1 delta cells. Moreover, TLC1 RNA specifically precipitated with epitope-tagged Est1p. These data indicate that Est1p is either a subunit of yeast telomerase or an accessory protein associated with telomerase that is essential in vitro for its activity.
Wright, JH, and VA Zakian. “
Protein-DNA interactions in soluble telosomes from Saccharomyces cerevisiae.”.
Nucleic Acids Res 23.9 (1995): ,
23, 9, 1454-60. Print.
AbstractTelomeric DNA in Saccharomyces is organized into a non-nucleosomal chromatin structure called the telosome that can be released from chromosome ends in soluble form by nuclease digestion (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210). The protein-DNA interactions of soluble telosomes were investigated by monitoring isolated telomeric DNA fragments for the retention of bound protein using both gel mobility shift and nitrocellulose filter-binding assays. Telosomal proteins remained associated with telomeric DNA at concentrations of ethidium bromide that dissociated nucleosomes. The protein-DNA interactions in the yeast telosome were also disrupted by much lower salt concentrations than those known to disrupt either the interactions of ciliate terminus-binding proteins with telomeric DNA or the interactions of histones with DNA in nucleosomes. Taken together, these data corroborate previously published nuclease mapping data indicating that telosomes are distinct in structure from conventional nucleosomes. These data also indicate that yeast do not possess telomere binding proteins similar to those detected in ciliates that remain tightly bound to telomeric DNA even in high salt. In addition, the characteristic gel mobility shift of soluble telosomes could be mimicked by complexes formed in vitro with yeast telomeric DNA and recombinant Rap1p suggesting that Rap1p, a known component of soluble yeast telosomes (Wright, J. H., Gottschling, D. E. and Zakian, V. A. (1992) Genes Dev. 6, 197-210; Conrad, M. N., Wright, J. H., Wolf, A. J. and Zakian, V. A. (1990) Cell 63, 739-750), is likely to be the major structural protein bound directly to yeast telomeric DNA.
Zakian, VA. “
Telomeres: beginning to understand the end.”.
Science 270.5242 (1995): ,
270, 5242, 1601-7. Print.
AbstractTelomeres are the protein-DNA structures at the ends of eukaryotic chromosomes. In yeast, and probably most other eukaryotes, telomeres are essential. They allow the cell to distinguish intact from broken chromosomes, protect chromosomes from degradation, and are substrates for novel replication mechanisms. Telomeres are usually replicated by telomerase, a telomere-specific reverse transcriptase, although telomerase-independent mechanisms of telomere maintenance exist. Telomere replication is both cell cycle- and developmentally regulated, and its control is likely to be complex. Because telomere loss causes the kinds of chromosomal changes associated with cancer and aging, an understanding of telomere biology has medical relevance.