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/MATα cells relative to MATa or MATα 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/MATα 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/α cells. Together, these results indicate that NHEJ regulation by mating type is achieved, at least in part, by a regulation of Lif2p activity

Dicentric breakage at telomere fusions

Nonhomologous end-joining (NHEJ) inhibition at telomeres ensures that native chromosome ends do not fuse together. But the occurrence and consequences of rare telomere fusions are not well understood. It is notably unclear whether a telomere fusion could be processed to restore telomere ends. Here we address the behavior of individual dicentrics formed by telomere fusion in the yeast Saccharomyces cerevisiae. Our approach was to first stabilize and amplify fusions between two chromosomes by temporarily inactivating one centromere. Next we analyzed dicentric breakage following centromere reactivation. Unexpectedly, dicentrics often break at the telomere fusions during progression through mitosis, a process that restores the parental chromosomes. This unforeseen result suggests a rescue pathway able to process telomere fusions and to back up NHEJ inhibition at telomeres

Mismatch tolerance by DNA Polymerase Pol4 in the course of nonhomologous end joining in Saccharomyces cerevisiae

In yeast, the nonhomologous end joining pathway (NHEJ) mobilizes the DNA polymerase Pol4 to repair DNA double-strand breaks when gap filling is required prior to ligation. Using telomere–telomere fusions caused by loss of the telomeric protein Rap1 and double-strand break repair on transformed DNA as assays for NHEJ between fully uncohesive ends, we show that Pol4 is able to extend a 3′-end whose last bases are mismatched, i.e., mispaired or unpaired, to the template strand

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

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 Rap 1 p 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

Multiple pathways inhibit NHEJ at telomeres

The nonhomologous end-joining (NHEJ) repair pathway is inhibited at telomeres, preventing chromosome fusion. In budding yeast Saccharomyces cerevisiae, the Rap1 protein directly binds the telomere sequences and is required for NHEJ inhibition. Here we show that the Rap1 C-terminal domain establishes two parallel inhibitory pathways through the proteins Rif2 and Sir4. In addition, the central domain of Rap1 inhibits NHEJ independently of Rif2 and Sir4. Thus, Rap1 establishes several independent pathways to prevent telomere fusions. We discuss a possible mechanism that would explain Rif2 multifunctionality at telomeres and the recent evolutionary origin of Rif2 from an origin recognition complex (ORC) subunit

The effect of the shortest telomere on cell proliferation

International audienceProgress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection

The effect of the shortest telomere on cell proliferation

International audienceProgress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection