Nathalie Grandin

Cdc13 Cooperates with the Yeast Ku Proteins and Stn1 To Regulate Telomerase Recruitment

ABSTRACT The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both onSTN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination.

Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13‐induced telomeric DNA damage in proliferating cells. Checkpoint‐deficient cdc13‐1 cells accumulated DNA damage and eventually senesced. However, these telomerase‐proficient cells could survive by using homologous recombination but, contrary to telomerase‐deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13‐1 mec3, as well as in telomerase‐deficient cdc13‐1 cells, which were Rad52‐ and Rad50‐dependent but Rad51‐independent, exclusively amplified the TG1–3 repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13‐1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13‐1–Ten1–Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase.

The Rad51 Pathway of Telomerase-Independent Maintenance of Telomeres Can Amplify TG1-3 Sequences in yku and cdc13 Mutants of Saccharomyces cerevisiae

ABSTRACT In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70Δ mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70Δ cells survived telomere deprotection by exclusively amplifying TG1-3 repeats (type II recombination). In a background lacking telomerase (tlc1Δ), this process predominated over type I recombination (amplification of subtelomeric Y′ sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70Δ or yku70Δ tlc1Δ mutants, but also in cdc13-1 YKU70+ tlc1Δ mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70Δ rad51Δ and cdc13-1 tlc1Δ rad51Δ cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG1-3 sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.

Activation of Mrc1, a mediator of the replication checkpoint, by telomere erosion

Background information. In budding yeast, the loss of either telomere sequences (in telomerase‐negative cells) or telomere capping (in mutants of two telomere end‐protection proteins, Cdc13 and Yku) lead, by distinct pathways, to telomeric senescence. After DNA damage, activation of Rad53, which together with Chk1 represents a protein kinase central to all checkpoint pathways, normally requires Rad9, a checkpoint adaptor.

Mitotic cyclins regulate telomeric recombination in telomerase-deficient yeast cells

ABSTRACT Telomerase-deficient mutants of Saccharomyces cerevisiae can survive death by senescence by using one of two homologous recombination pathways. The Rad51 pathway amplifies the subtelomeric Y′ sequences, while the Rad50 pathway amplifies the telomeric TG1-3 repeats. Here we show that telomerase-negative cells require Clb2 (the major B-type cyclin in this organism), in association with Cdc28 (Cdk1), to generate postsenescence survivors at a normal rate. The Rad50 pathway was more sensitive to the absence of Clb2 than the Rad51 pathway. We also report that telomerase RAD50 RAD51 triple mutants still generated postsenescence survivors. This novel Rad50- and Rad51-independent pathway of telomeric recombination also appeared to be controlled by Clb2. In telomerase-positive cells, a synthetic growth defect between mutations in CLB2 and RAD50 or in its partners in the conserved MRX complex, MRE11 and XRS2, was observed. This genetic interaction was independent of Mre11 nuclease activity but was dependent on a DNA repair function. The present data reveal an unexpected role of Cdc28/Clb2 in telomeric recombination during telomerase-independent maintenance of telomeres. They also uncover a functional interaction between Cdc28/Clb2 and MRX during the control of the mitotic cell cycle.

The Cdc14 phosphatase is functionally associated with the Dbf2 protein kinase in Saccharomycescerevisiae

Abstract The Saccharomyces cerevisiae Cdc14 protein phosphatase and Dbf2 protein kinase have been implicated to act during late M phase, but their functions are not known. We report here that CDC14 is a low-copy suppressor of the dbf2-2 mutation at 37° C. The kinase activity of Dbf2 accumulated at a high level, in vivo, during a cdc14 arrest and was also much higher in cdc14 mutant cells at the permissive temperature of growth, therefore in cycling mutant cells than in cycling wild-type cells. This correlated with the accumulation of the more slowly migrating form of Dbf2, previously shown to correspond to the hyperphosphorylated form of the protein. The finding that the dbf2-2 mutation could be rescued following overproduction of catalytically inactive forms of Cdc14 suggested that the control of Dbf2 activity by Cdc14 might be only indirect and independent of Cdc14 phosphatase activity. However, it was found that Cdc14 could form oligomers within the cell, thus leaving open the possibility that catalytically inactive Cdc14 might associate with wild-type Cdc14 and rescue dbf2-2 in a phosphatase-dependent manner. We confirmed that overexpression of CDC14 could rescue mutations in CDC15, which encodes another kinase also implicated to act in late M phase. Cells of a cdc15-2dbf2-2 double mutant died at temperatures much lower than did either single mutant, whereas there was only a slight additive phenotype in the cdc14-1 dbf2-2 and cdc14-1 cdc15-2 double mutant cells. Finally, functional association between Cdc14 and Dbf2 (and also Cdc15) was confirmed by the finding that the cdc14, dbf2 and cdc15 mutations could be partially rescued by the addition of 1.2 M sorbitol to the culture medium. Our data are the first to demonstrate a functional link between Cdc14 and Dbf2 based on both biochemical and genetic information.

Telomerase- and Rad52-Independent Immortalization of Budding Yeast by an Inherited-Long-Telomere Pathway of Telomeric Repeat Amplification

ABSTRACT In the absence of telomerase, telomeres erode, provoking accumulation of DNA damage and death by senescence. Rare survivors arise, however, due to Rad52-based amplification of telomeric sequences by homologous recombination. The present study reveals that in budding yeast cells, postsenescence survival relying on amplification of the TG1-3 telomeric repeats can take place in the absence of Rad52 when overelongated telomeres are present during senescence (hence its designation ILT, for inherited-long-telomere, pathway). By growth competition, the Rad52-independent pathway was almost as efficient as the Rad51- and Rad52-dependent pathway that predominates in telomerase-negative cells. The ILT pathway could also be triggered by increased telomerase accessibility before telomerase removal, combined with loss of telomere protection, indicating that prior accumulation of recombination proteins was not required. The ILT pathway was dependent on Rad50 and Mre11 but not on the Rad51 recombinase and Rad59, thus making it distinct from both the type II (budding yeast ALT [alternative lengthening of telomeres]) and type I pathways amplifying the TG1-3 repeats and subtelomeric sequences, respectively. The ILT pathway also required the Rad1 endonuclease and Elg1, a replication factor C (RFC)-like complex subunit, but not Rad24 or Ctf18 (two subunits of two other RFC-like complexes), the Dnl4 ligase, Yku70, or Nej1. Possible mechanisms for this Rad52-independent pathway of telomeric repeat amplification are discussed. The effects of inherited long telomeres on Rad52-dependent recombination are also reported.

Mrc1, a non-essential DNA replication protein, is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase.

Proteins involved in telomere end protection have previously been identified. In Saccharomyces cerevisiae, Cdc13, Yku and telomerase, mainly, prevent telomere uncapping, thus providing telomere stability and avoiding degradation and death by senescence. Here, we report that in the absence of Mrc1, a component of the replication forks, telomeres of cdc13 or yku70 mutants exhibited increased degradation, while telomerase-negative cells displayed accelerated senescence. Moreover, deletion of MRC1 increased the single-strandedness of the telomeres in cdc13-1 and yku70Δ mutant strains. An mrc1 deletion strain also exhibited slight but stable telomere shortening compared to a wild-type strain. Loss of Mrc1’s checkpoint function alone did not provoke synthetic growth defects in combination with the cdc13-1 mutation. Combinations between the cdc13-1 mutation and deletion of either TOF1 or PSY2, coding for proteins physically interacting with Mrc1, also resulted in a synthetic growth defect. Thus, the present data suggest that non-essential components of the DNA replication machinery, such as Mrc1 and Tof1, may have a role in telomere stability in addition to their role in fork progression.

Budding yeast 14-3-3 proteins contribute to the robustness of the DNA damage and spindle checkpoints

Cells respond to DNA or mitotic spindle damage by activating specific pathways that halt the cell cycle to allow for possible repair. Here, we report that inactivation of one of the Saccharomyces cerevisiae 14-3-3 proteins, Bmh1, as well as the bmh1-S189P bmh2 mutant, failed to exhibit normal spindle damage-induced cell cycle delay and conferred hypersensitivity to benomyl or nocodazole. These defects were additive with those conferred by the bub2 and mad2 spindle checkpoint mutations. Following cdc13-1-induced DNA damage, the 14-3-3 response was additive with those provided by the Mec1 (ATR-related)-controlled Rad53 (CHK2-related) and Chk1 (CHK1-related) checkpoint pathways and also distinct from the PKA (Protein Kinase A)-controlled response. Therefore, the budding yeast 14-3-3 proteins contribute to the robustness of the two major mitotic checkpoints and, by doing so, may also ensure optimal coordination between the responses to two distinct types of damage.

Rvb2/reptin physically associates with telomerase in budding yeast

Est2 physically interacts with Rvb2 by anti tag coimmunoprecipitation (View interaction)