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A J Lustig

Publications and source records attributed to A J Lustig.

At least 19 recordsLinked to original sources
PubMed2001

Intrachromatid excision of telomeric DNA as a mechanism for telomere size control in Saccharomyces cerevisiae.

We have previously identified a process in the yeast Saccharomyces cerevisiae that results in the contraction of elongated telomeres to wild-type length within a few generations. We have termed this process telomeric rapid deletion (TRD). In this study, we use a combination of physical and genetic assays to investigate the mechanism of TRD. First, to distinguish among several recombinational and nucleolytic pathways, we developed a novel physical assay in which HaeIII restriction sites are positioned within the telomeric tract. Specific telomeres were subsequently tested for HaeIII site movement between telomeres and for HaeIII site retention during TRD. Second, genetic analyses have demonstrated that mutations in RAD50 and MRE11 inhibit TRD. TRD, however, is independent of the Rap1p C-terminal domain, a central regulator of telomere size control. Our results provide evidence that TRD is an intrachromatid deletion process in which sequences near the extreme terminus invade end-distal sequences and excise the intervening sequences. We propose that the Mre11p-Rad50p-Xrs2p complex prepares the invading telomeric overhang for strand invasion, possibly through end processing or through alterations in chromatin structure.

Chromatids↗
PubMed2000

Telomere structure regulates the heritability of repressed subtelomeric chromatin in Saccharomyces cerevisiae.

Telomeres, the protein-DNA structures present at the termini of linear chromosomes, are capable of conferring a reversible repression of Pol II- and Pol III-transcribed genes positioned in adjacent subtelomeric regions. This phenomenon, termed telomeric silencing, is likely to be the consequence of a more global telomere position effect at the level of chromatin structure. To understand the role of telomere structure in this position effect, we have developed an assay to distinguish between the heritability of transcriptionally repressed and derepressed states in yeast. We have previously demonstrated that an elongated telomeric tract leads to hyperrepression of telomere-adjacent genes. We show here that the predominant effect of elongated telomeres is to increase the inheritance of the repressed state in cis. Interestingly, the presence of elongated telomeres overcomes the partial requirement of yCAF-1 in silencing. We propose that the formation of a specific telomeric structure is necessary for the heritability of repressed subtelomeric chromatin.

Chromatin↗
PubMed1998

The yeast Ku heterodimer is essential for protection of the telomere against nucleolytic and recombinational activities.

The Ku heterodimer, conserved in a wide range of eukaryotes, plays a multiplicity of roles in yeast. First, binding of Ku, which is composed of a 70 kDa (Hdf1p) and an 80 kDa (Hdf2p) subunit [1-3], to double-strand breaks promotes non-homologous end-to-end joining of DNA [3]. Second, Ku appears to participate in DNA replication, regulating both the number of rounds of replication permissible within the cell cycle and the structure of the initiation complex [3,4]. Furthermore, mutations in HDF1 or HDF2 rapidly reduce telomeric poly (TG1-3) tract size [1-3], hinting also at a possible telomeric function of Ku. We show here that the two subunits of the Ku heterodimer play a key role in maintaining the integrity of telomere structure. Mutations in either Ku subunit increased the single-strandedness of the telomere in a cell-cycle-independent fashion, unlike wild-type cells which form 3' poly(TG1-3) overhangs exclusively in late S phase [5]. In addition, mutations enhanced the instability of elongated telomeres to degradation and recombination. Both Ku subunits genetically interacted with the putative single-stranded telomere-binding protein Cdc13p. We propose that Ku protects the telomere against nucleases and recombinases.

Antigens, Nuclear↗
PubMed1998

DNA dynamics: different means to a common end?

A ribosomal frameshift is required for the synthesis of an essential component of the yeast telomerase pathway; this and other findings on telomerases from many species raise interesting questions regarding the evolutionary relationship between telomerases and retrotransposons lacking long terminal repeats.

Animals↗
PubMed1998

Mechanisms of silencing in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, heterochromatin-like regions are formed at the silent mating type loci and at telomeres. The past year of investigations has led to a clearer understanding of the nature of nucleation and spreading of heterochromatin, as well as uncovering a fascinating link between silencing, the nucleolus and aging.

Animals↗
PubMed1998

Sir3p domains involved in the initiation of telomeric silencing in Saccharomyces cerevisiae.

Previous studies from our laboratory have demonstrated that tethering of Sir3p at the subtelomeric/telomeric junction restores silencing in strains containing Rap1-17p, a mutant protein unable to recruit Sir3p. This tethered silencing assay serves as a model system for the early events that follow recruitment of silencing factors, a process we term initiation. A series of LexA fusion proteins in-frame with various Sir3p fragments were constructed and tested for their ability to support tethered silencing. Interestingly, a region comprising only the C-terminal 144 amino acids, termed the C-terminal domain (CTD), is both necessary and sufficient for restoration of silencing. Curiously, the LexA-Sir3(N205) mutant protein overcomes the requirement for the CTD, possibly by unmasking a cryptic initiation site. A second domain spanning amino acids 481-835, termed the nonessential for initiation domain (NID), is dispensable for the Sir3p function in initiation, but is required for the recruitment of the Sir4p C terminus. In addition, in the absence of the N-terminal 481 amino acids, the NID negatively influences CTD activity. This suggests the presence of a third region, consisting of the N-terminal half (1-481) of Sir3p, termed the positive regulatory domain (PRD), which is required to initiate silencing in the presence of the NID. These data suggest that the CTD "active" site is under both positive and negative control mediated by multiple Sir3p domains.

DNA-Binding Proteins↗
PubMed1996

A novel mechanism for telomere size control in Saccharomyces cerevisiae.

One of the central requirements for eukaryotic chromosome stability is the maintenance of the simple sequence tracts at telomeres. In this study, we use genetic and physical assays to reveal the nature of a novel mechanism by which telomere length is controlled. This mechanism, telomeric rapid deletion (TRD), is capable of reducing elongated telomeres to wild-type tract length in an apparently single-division process. The deletion of telomeres to wild-type lengths is stimulated by the hpr1 mutation, suggesting that TRD in these cells is the consequence of an intrachromatid pathway. Paradoxically, TRD is also dependent on the lengths of the majority of nonhomologous telomeres in the cell. Defects in the chromatin-organizing protein Sir3p increase the rate of hpr1-induced rapid deletion and specifically change the spectrum of rapid deletion events. We propose a model in which interactions among telosomes of nonhomologous chromosomes form higher order complexes that restrict the access of the intrachromatid recombination machinery to telomeres. This mechanism of size control is distinct from that mediated through telomerase and is likely to maintain telomere length within a narrow distribution.

Chromosome Deletion↗
PubMed1996

Genetic analysis of Rap1p/Sir3p interactions in telomeric and HML silencing in Saccharomyces cerevisiae.

We have identified three SIR3 suppressors of the telomeric silencing defects conferred by missense mutations within the Rap1p C-terminal tail domain (aa 800-827). Each SIR3 suppressor was also capable of suppressing a rap1 allele (rap1-21), which deletes the 28 aa C-terminal tail domain, but none of the suppressors restored telometric silencing to a 165 amino acid truncation allele. These data suggest a Rap1p site for Sir3p association between the two truncation points (aa 664-799). In SIR3 suppressor strains lacking the Rap1p C-terminal tail domain, the presence of a second intragenic mutation within the rap1s domain (aa 727-747), enhanced silencing 30-300-fold. These data suggest a competition between Sir3p and factors that interfere with silencing for association in the rap1s domain. Rap1-21 strains containing both wild-type Sir3p and either of the Sir3 suppressor proteins displayed a 400-4000-fold increase in telomeric silencing over rap1-21 strains carrying either Sir3p suppressor in the absence of wild-type Sir3p. We propose that this telomere-specific synergism is mediated in part through stabilization of Rap1p/Sir3p telometric complexes by Sir3p-Sir3p interactions.

Alleles↗
PubMed1996

Tethered Sir3p nucleates silencing at telomeres and internal loci in Saccharomyces cerevisiae.

Rap1p binds to sites embedded within the Saccharomyces cerevisiae telomeric TG1-3 tract. Previous studies have led to the hypothesis that Rap1p may recruit Sir3p and Sir3p-associating factors to the telomere. To test this, we tethered Sir3p adjacent to the telomere via LexA binding sites in the rap1-17 mutant that truncates the Rap1p C-terminal 165 amino acids thought to contain sites for Sir3p association. Tethering of LexA-Sir3p adjacent to the telomere is sufficient to restore telomeric silencing, indicating that Sir3p can nucleate silencing at the telomere. Tethering of LexA-Sir3p or the LexA-Sir3p(N2O5) gain-of-function protein to a telomeric LexA site hyperrepresses an adjacent ADE2 gene in wild-type cells. Hence, Sir3p recruitment to the telomere is limiting in telomeric silencing. In addition, LexA-Sir3p(N2O5) hyperrepresses telomeric silencing when tethered to a subtelomeric site 3.6 kb from the telomeric tract. This hyperrepression is dependent on the C terminus of Rap1p, suggesting that subtelomeric LexA-Sir3p(N205) can interact with Rap1p-associated factors at the telomere. We also demonstrate that LexA-Sir3p or LexA-Sir3p(N205) tethered in cis with a short tract of telomeric TG1-3 sequences is sufficient to confer silencing at an internal chromosomal position. Internal silencing is enhanced in rap1-17 strains. We propose that sequestration of silencing factors at the telomere limits the efficiency of internal silencing.

Bacterial Proteins↗
PubMed1995

The carboxy termini of Sir4 and Rap1 affect Sir3 localization: evidence for a multicomponent complex required for yeast telomeric silencing.

The Silent Information Regulatory proteins, Sir3 and Sir4, and the telomeric repeat-binding protein RAP1 are required for the chromatin-mediated gene repression observed at yeast telomeric regions. All three proteins are localized by immunofluorescence staining to foci near the nuclear periphery suggesting a relationship between subnuclear localization and silencing. We present several lines of immunological and biochemical evidence that Sir3, Sir4, and RAP1 interact in intact yeast cells. First, immunolocalization of Sir3 to foci at the yeast nuclear periphery is lost in rap1 mutants carrying deletions for either the terminal 28 or 165 amino acids of RAP1. Second, the perinuclear localization of both Sir3 and RAP1 is disrupted by overproduction of the COOH terminus of Sir4. Third, overproduction of the Sir4 COOH terminus alters the solubility properties of both Sir3 and full-length Sir4. Finally, we demonstrate that RAP1 and Sir4 coprecipitate in immune complexes using either anti-RAP1 or anti-Sir4 antibodies. We propose that the integrity of a tertiary complex between Sir4, Sir3, and RAP1 is involved in both the maintenance of telomeric repression and the clustering of telomeres in foci near the nuclear periphery.

Cell Compartmentation↗
PubMed1994

Mutational analysis defines a C-terminal tail domain of RAP1 essential for Telomeric silencing in Saccharomyces cerevisiae.

Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing.

Alleles↗
PubMed1993

RAP1 and telomere structure regulate telomere position effects in Saccharomyces cerevisiae.

To investigate the role of the yeast telomere-, silencing-, and UAS-binding protein RAP1 in telomere position effects, we have characterized two sets of mutant cells: (1) a set of rap1 alleles (termed the rap1t alleles) that produce truncated RAP1 proteins missing the carboxy-terminal 144-165 amino acids; and (2) null mutants of the RIF1 gene, encoding a protein capable of interaction with the carboxyl terminus of RAP1. The data presented here indicate that loss of the carboxyl terminus of RAP1 abolishes position effects at yeast telomeres and diminishes silencing at the HML locus. Elimination of position effects in these cells is associated with increased accessibility to the Escherichia coli dam methylase in vivo. Thus, the carboxy-terminal domain of RAP1 is required for telomere position effects. In contrast, rif1 deletion alleles increase the frequency of repressed cells. Using the rap1t alleles to generate wild-type cells differing only in telomere tract lengths, we also show that telomere position effects are highly sensitive to changes in the size (or structure) of the telomeric tract. Longer poly(G1-3T) tracts can increase the frequency of transcriptional repression at the telomere, suggesting that telomeric poly(G1-3T) tracts play an active role in the formation or stability of subtelomeric transcriptional states.

Chromatin↗
PubMed1992

Hoogsteen G-G base pairing is dispensable for telomere healing in yeast.

The G-rich strands of most eukaryotic telomeres are capable of forming highly folded structures in vitro, mediated, in part, through Hoogsteen G-G base pairing. The ability of most telomeres to form these structures has led to the suggestion that they play an important role in telomere addition. I have investigated this possibility in the yeast Saccharomyces cerevisiae through the use of an in vivo assay that measures healing via poly(G1-3T) addition onto plasmid substrates containing synthetic telomeres. Synthetic telomere healing is a highly size- and sequence-specific process that allows the discrimination of telomeres of differing efficiency. Plasmids containing synthetic telomeres with differing abilities to form secondary structures were tested in this assay for healing in vivo. The results of this study demonstrate that telomeres incapable of forming Hoogsteen base pairs nonetheless serve as efficient substrates for poly(G1-3T) addition, indicating that intramolecular Hoogsteen G-G base pairing is not essential for this process.

Base Composition↗