Rap1 prevents telomere fusions by nonhomologous end joining

Abstract

Telomeres protect chromosomes from end-to-end fusions. In yeast Saccharomyces cerevisiae, the protein Rap1 directly binds telomeric DNA. Here, we use a new conditional allele of RAP1 and show that Rap1 loss results in frequent fusions between telomeres. Analysis of the fusion point with restriction enzymes indicates that fusions occur between telomeres of near wild-type length. Telomere fusions are not observed in cells lacking factors required for nonhomologous end joining (NHEJ), including Lig4 (ligase IV), KU and the Mre11 complex. SAE2 and TEL1 do not affect the frequency of fusions. Together, these results show that Rap1 is essential to block NHEJ between telomeres. Since the presence of Rap1 at telomeres has been conserved through evolution, the establishment of NHEJ suppression by Rap1 could be universal.

Figure 1

Rap1 loss in rap1-(Δ) cells progressing toward stationary phase. (A) Growth curve of wild-type and rap1-(Δ) cells in rich medium at 30°C. Yeast strains ZMY60 (wild type) and Lev391 (rap1-(Δ)) were maintained in exponential phase by successive dilutions in rich medium for 2 days and at time 0 allowed to exhaust the medium. The doubling time is estimated from the initial exponential growth. (B) Immunoblot showing Rap1 level in wild-type and rap1-(Δ) cells. Rap1 in rap1-(Δ) cells is tagged with four HA epitopes. To detect Rap1 and Rap1 tagged with HA epitopes, we used, respectively, a rabbit polyclonal antibody directed against the carboxy-terminal region of Rap1 (α-Rap1) and a monoclonal antibody directed against the HA epitope (α-HA). Loading was controlled by gel staining.

Figure 2

Telomere fusions in rap1-(Δ) cells progressing toward stationary phase. (A) Schematic representation of X and Y′ telomeres in S. cerevisiae and relative positions of the primers used for PCR amplification. In the sequenced yeast genome, there are 15 X telomeres and 17 Y′ telomeres. The fusion of two telomeres should give a PCR product of about 1000 bp plus the TG_1–3 telomeric repeats at the junction. Fusions between two X or two Y′ telomeres form quasiperfect palindromes and are unlikely to be amplified. (B) Detection by PCR of telomere fusions in rap1-(Δ) cells reaching stationary phase. Time points are the same as in Figure 1. (C) Complementation of rap1-(Δ) in Lev391 with a wild-type copy of RAP1 integrated at URA3. Cells were grown to saturation in rich medium for 5 days. Telomere fusions are not amplified with only one of the two primers X or Y′. (D) Schematic representation of PCR amplification, cloning and restriction analysis of telomere fusions.

Figure 3

Telomere fusions among cells with RAP1 on a plasmid. (A) Schematic representation of the yeast strains deleted for RAP1 and carrying a wild-type copy of RAP1 either on a plasmid or integrated in a chromosome. (B) Continuous loss of wild-type RAP1 gene on a plasmid causes telomere fusions. In the yeast strain Lev9, plasmid pCEN-Sup4-RAP1 was replaced by centromeric plasmid pRS316-RAP1 or by pRS306-RAP1 integrated at URA3. The control RAP1 strain is W303-1a. Cells were grown to saturation in rich medium for 5 days. Telomere fusions were amplified by PCR. (C) Relative quantification of telomere fusions. Strains Lev9 shuffled with pRS316-RAP1 and Lev391 (rap1-(Δ)) were grown to saturation in rich medium for 5 days. Fusions were amplified by PCR and quantified as described in Materials and methods.

Figure 4

NHEJ factors are required for telomere fusions. (A) Yeast strains ZMY60 (wild type), Lev391 (rap1-(Δ)), Lev397 (rap1-(Δ) yku70-Δ), Ybp43 (rap1-(Δ) yku80-Δ), Ybp11 (rap1-(Δ) lig4-Δ), Lev396 (rap1-(Δ) lif1-Δ) and Ybp14 (rap1-(Δ) lif2-Δ) were grown to saturation in rich medium for 7 days. Strains ZMY60, Lev391 and Ybp14 transformed with plasmid pRS314 and strain Ybp14 transformed with plasmid pRS314-LIF2 were grown to saturation in synthetic medium lacking tryptophan for 5 days. Telomere fusions were amplified by PCR. (B) Yeast strains ZMY60, Lev391, Ybp29 (rap1-(Δ) mre11-Δ), Ybp27 (rap1-(Δ) rad50-Δ), Ybp9 (rap1-(Δ) xrs2-Δ), Ybp40 (sae2-Δ), Ybp41 (tel1-Δ), Ybp31 (rap1-(Δ) sae2-Δ) and Ybp23 (rap1-(Δ) tel1-Δ) were grown to saturation in rich medium for 7 days. Telomere fusions were amplified by PCR.

Figure 5

Quantification of telomere fusions. (A) Schematic map of the pRS316-Fusion-Long and pRS316-Fusion-Short plasmids. In pRS316-Fusion-Long, the X element comes from the right end of chromosome II. An ApaLI site is present at the junction and the two TG_1–3 repeats pointing at each other are about 350 and 270 bp. In pRS316-Fusion-Short, the X element comes from the right end of chromosome IX. The sum of the TG_1–3 inverted repeats is about 200 bp. pRS316-Fusion-Long and pRS316-Fusion-Short were separately reintroduced into wild-type cells. (B) Quantification of fusions between X and Y′ telomeres. Yeast strains ZMY60 (wild type) transformed with pRS316-Fusion-Long and pRS316-Fusion-Short were grown to saturation for 5 days in medium lacking uracil. Lev391 (rap1-(Δ)), Ybp31 (rap1-(Δ) sae2-Δ) and Ybp23 (rap1-(Δ) tel1-Δ) were grown to saturation for 5 days in rich medium. Fusions were amplified by PCR and quantified as described in Materials and methods. (C) Number of fusions per genome. Plasmid copy number was estimated by Southern analysis (data not shown). The signals from strains Lev391 (rap1-(Δ)) and Ybp31 (rap1-(Δ) sae2-Δ) were normalized with the signals from strain ZMY60 (wild type) transformed with pRS316-Fusion-Long. The signals from strain Ybp23 (rap1-(Δ) tel1-Δ) were normalized with the signals from strain ZMY60 (wild type) transformed with pRS316-Fusion-Short. Mean and standard deviation were calculated by averaging 12 amplifications (three dilutions of four independent samples).

Figure 6

Telomere fusions reduce cell viability. (A) Schematic representation of single cell micromanipulation during its first division. Yeast strains were grown to saturation in rich medium for 3–4 weeks. Cells were spread onto plates, micromanipulated onto a grid and left at 30°C. The two products of the first division were separated as budded cells and placed adjacent to each other on the same line. An example has been recorded at each step and is shown. (B) After micromanipulation, cells were grown for 4 days at 30°C. Possible outcomes are shown. Dashed rectangles denote the cell that gave rise to a viable colony, the dashed circle denotes the initial cell that failed to restart growth or achieve its first division and dashed stars denote the cell that failed to achieve its first division or formed an unviable microcolony of two to a few hundred cells. (C) Morphology of cells that failed to form a viable colony. (D) Cell outcomes after stationary phase. Yeast strains ZMY60 (wild type), Lev391 (rap1-(Δ)), Lev396 (rap1-(Δ) lif1-Δ) and Lev398 (lif1-Δ) were grown and micromanipulated as described in panel A. The data come from two independent experiments (n_total=150 cells). P-values were calculated using a Student's test.