Biomedical subjects

S Marcand

Publications and source records attributed to S Marcand.

9 recordsLinked to original sources

PubMed2001

NHEJ regulation by mating type is exercised through a novel protein, Lif2p, essential to the ligase IV pathway.

In the yeast Saccharomyces cerevisiae, DNA double strand break (DSB) repair by nonhomologous end-joining (NHEJ) requires the DNA end-binding heterodimer Yku70p-Yku80p and the ligase Dnl4p associated with its cofactor Lif1p. NHEJ efficiency is down-regulated in MATa/MATalpha cells relative to MATa or MATalpha cells, but the mechanism of this mating type regulation is unknown. Here we report the identification of Lif2p, a S. cerevisiae protein that interacts with Lif1p in a two-hybrid system. Disruption of LIF2 abolishes the capacity of cells to repair DSBs by end-joining to the same extent than lif1 and dnl4 mutants. In MATa/MATalpha cells, Lif2p steady-state level is strongly repressed when other factors involved in NHEJ are unaffected. Increasing the dosage of the Lif2p protein can suppress the NHEJ defect in a/alpha cells. Together, these results indicate that NHEJ regulation by mating type is achieved, at least in part, by a regulation of Lif2p activity.

Antigens, Nuclear↗

PubMed2000

Cell cycle restriction of telomere elongation.

Telomere elongation by telomerase balances the progressive shortening of chromosome ends due to the succession of replication cycles [1] [2]. Telomerase activity is regulated in vivo at its site of action by the telomere itself. In yeast and human cells, the mean telomere length is maintained at a constant value through a cis-inhibition of telomerase by factors specifically bound to the telomeric DNA [3] [4] [5] [6] [7]. Here, we address an unexplored aspect of telomerase regulation by testing the link between telomere dynamics and cell cycle progression in the budding yeast Saccharomyces cerevisiae. We followed the elongation of an abnormally shortened telomere and observed that, like telomere shortening in the absence of telomerase, telomere elongation is linked to the succession of cell divisions. In cells progressing synchronously through the cell cycle, telomere elongation coincided with the time of telomere replication. On a minichromosome, a replication defect partially suppressed telomere elongation, suggesting a coupling between in vivo telomerase activity and conventional DNA replication.

Cell Cycle↗

PubMed1999

Progressive cis-inhibition of telomerase upon telomere elongation.

In yeast, the constant length of telomeric DNA results from a negative regulation of telomerase by the telomere itself. Here we follow the return to equilibrium of an abnormally shortened telomere. We observe that telomere elongation is restricted to a few base pairs per generation and that its rate decreases progressively with increasing telomere length. In contrast, in the absence of telomerase or in the presence of an over-elongated telomere, the degradation rate linked to the succession of generations appears to be constant, i.e. independent of telomere length. Together, these results indicate that telomerase is gradually inhibited at its site of action by the elongating telomere. The implications of this finding for the dynamics of telomere length regulation are discussed in this study.

Alleles↗

PubMed1997

A protein-counting mechanism for telomere length regulation in yeast.

In the yeast Saccharomyces cerevisiae, telomere elongation is negatively regulated by the telomere repeat-binding protein Rap1p, such that a narrow length distribution of telomere repeat tracts is observed. This length regulation was shown to function independently of the orientation of the telomere repeats. The number of repeats at an individual telomere was reduced when hybrid proteins containing the Rap1p carboxyl terminus were targeted there by a heterologous DNA-binding domain. The extent of this telomere tract shortening was proportional to the number of targeted molecules, consistent with a feedback mechanism of telomere length regulation that can discriminate the precise number of Rap1p molecules bound to the chromosome end.

Binding Sites↗

PubMed1997

Rap1p and telomere length regulation in yeast.

Telomere length in the yeast Saccharomyces cerevisiae is under stringent genetic control such that a narrow length distribution of TG1-3 repeats is observed. Previous studies have shown that Rap1p, which binds to the double-stranded telomeric repeats, plays a role in regulating repeat length: point mutations in the Rap1p C-terminus often result in a higher average telomere length and deletion of this region causes extreme telomere elongation. We have investigated further the role of Rap1p in this process. Our results suggest that telomere length is regulated by a negative feedback mechanism that can sense the number of Rap1p molecules bound at the chromosome end. This length regulatory mechanism requires two other proteins, Rif1p and Rif2p, that interact with each other and with the Rap1p C-terminus. Although the same C-terminal domain of Rap1p is also involved in the initiation of telomere position effect (telomeric transcriptional silencing), this Rap1p function appears to be separate from, and indeed antagonistic to, its role in telomere length regulation.

Carrier Proteins↗

PubMed1996

Chromatin: a sticky silence.

Recent findings indicate that heterochromatin serves as a molecular sink for factors involved in chromatin-mediated repression of gene expression; long-range interactions that position a euchromatic gene near a heterochromatin domain influence its susceptibility to transcriptional silencing.

Animals↗

PubMed1996

Evidence for silencing compartments within the yeast nucleus: a role for telomere proximity and Sir protein concentration in silencer-mediated repression.

Transcriptional repression at the silent mating-type loci in yeast requires the targeting of silent information regulator (Sir) proteins through specific interactions formed at cis-acting silencer elements. We show here that a reporter gene flanked by two functional silencers is not repressed when integrated at >200 kb from a telomere. Repression is restored by creation of a new telomere 13 kb from the integrated reporter or by elevated expression of SIR1, SIR3, and/or SIR4. Coupled expression represses in an additive manner, suggesting that all three factors are in limiting concentrations. When overexpressed, Sir3 and Sir4 are dispersed throughout the nucleoplasm, in contrast to wild-type cells where they are clustered in a limited number of foci together with telomeres. Efficient silencer function thus seems to require either proximity to a pool of concentrated Sir proteins, that is, proximity to telomeres, or delocalization of the silencing factors.

Base Sequence↗

PubMed1996

Silencing of genes at nontelomeric sites in yeast is controlled by sequestration of silencing factors at telomeres by Rap 1 protein.

Rap1p binds to silencer elements and telomeric repeats in yeast, where it appears to initiate silencing by recruiting Sir3p and Sir4p to the chromosome through interactions with its carboxy-terminal domain. Sir3p and Sir4p interact in vitro with histones H3 and H4 and are likely to be structural components of silent chromatin. We show that targeting of these Sir proteins to the chromosome is sufficient to initiate stable silencing either at a silent mating-type locus lacking a functional silencer element or at a telomere in a strain in which the Rap1p carboxy-terminal silencing domain has been deleted. Silencing by Sir protein targeting can also be initiated at a telomere-proximal site (ADH4), but is much weaker at an internal chromosomal locus (LYS2). Strikingly, deletion of the Rap1p silencing domain, which abolishes telomeric silencing, improves targeted silencing at LYS2 by both Sir3p and Sir4p, while weakening the silencing activity of these proteins at or near a telomere. This effect may result from the release of Sir proteins from the telomeres, thus increasing their effective concentration at other chromosomal sites. We suggest that telomeres and Rap1p serve a regulatory role in sequestering Sir proteins at telomeres, controlling silencing at other loci in trans and preventing indiscriminate gene silencing throughout the genome.

Chromosomes, Fungal↗

PubMed1996

Cooperation at a distance between silencers and proto-silencers at the yeast HML locus.

Transcriptional repression at the silent yeast mating type loci is achieved through the formation of a particular nucleoprotein complex at specific cis-acting elements called silencers. This complex in turn appears to initiate the spreading of a histone binding protein complex into the surrounding chromatin, which restricts accessibility of the region to the transcription machinery. We have investigated long-range, cooperative effects between silencers by studying the repression of a reporter gene integrated at the HML locus flanked by various combinations of wild-type and mutated silencer sequences. Two silencers can cooperate over >4000 bp to repress transcription efficiently. More importantly, a single binding site for either the repressor activator protein 1 (Rap1), the autonomous replicating sequence (ARS) binding factor 1 (Abf1) or the origin recognition complex (ORC) can enhance the action of a distant silencer without acting as a silencer on its own. Functional cooperativity is demonstrated using a quantitative assay for repression, and varies with the affinity of the binding sites used. Since the repression mechanism is Sir dependent, the Rap1, ORC and/or Abf1 proteins bound to distant DNA elements may interact to create an interface of sufficiently high affinity such that Sir-containing complexes bind, nucleating the silent chromatin state.

Base Sequence↗