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, 24 (17), 3128-35

Taz1, Rap1 and Rif1 Act Both Interdependently and Independently to Maintain Telomeres

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Taz1, Rap1 and Rif1 Act Both Interdependently and Independently to Maintain Telomeres

Kyle M Miller et al. EMBO J.

Abstract

Telomere protection and maintenance are accomplished through the coordinated actions of telomere-specific DNA binding proteins and their interacting partners. The fission yeast ortholog of human TRF1/2, Taz1, binds telomeric DNA and regulates numerous aspects of telomere function. Here, we ask which aspects of Taz1 function are mediated through its interacting proteins, Rap1 and Rif1. We demonstrate that rap1+ deletion phenocopies some, but not all, aspects of taz1Delta telomere dysfunction, while Rif1 exhibits a very different functional spectrum. Rap1 acts in a Taz1-dependent pathway to prevent chromosome end fusions and regulate telomeric 3' overhang formation, while Rif1 is dispensable for these functions. Telomerase inhibition by Taz1 is mediated by two separate pathways, one involving Rap1 and the other involving Rif1. In contrast, Taz1 is uniquely required to prevent chromosomal entanglements and missegregation at cold temperatures. Strikingly, while rap1+ deletion exacerbates the cold sensitivity of taz1Delta cells, rif1+ deletion restores full viability. Thus, Rap1 and Rif1 are each required for a subset of the functions of Taz1, but each acquires Taz1-independent functions in its absence. Furthermore, Taz1 can function independently of its known binding partners.

Figures

Figure 1
Figure 1
Analysis of telomere length and 3′ telomeric overhang formation in various telomere mutants. (A) Telomere length. Genomic DNA was digested with Apa1, resolved on a 1% agarose gel, blotted and hybridized with a telomeric probe. (B) 3′ telomeric overhangs. EcoR1-digested genomic DNA was resolved on a 1% agarose gel and then hybridized under nondenaturing or denaturing conditions in duplicate gels to a G-strand-specific telomere probe. Due to the fragment size requirements for in-gel hybridization, wt DNA samples were digested with HindIII so that the terminal telomere fragments would be large enough for analysis (Tomita et al, 2003); note that the HindIII sites are ∼6 kb proximal to the EcoR1 sites on each chromosome.
Figure 2
Figure 2
Roles of Rap1 and Rif1 in preventing telomere fusions. (A) Map of fission yeast chromosomes showing telomeric Not1 restriction fragments. Digestion with NotI releases fragments L and I from Chr I and fragments M and C from Chr II. Chr III lacks Not1 sites. (B) PFGE of NotI-digested genomic DNA, blotted and hybridized with a telomere probe. The asterisk denotes the mobility of fragment C as well as any C-containing telomere fusion, which are not resolvable under these conditions.
Figure 3
Figure 3
Analysis of fission yeast telomere mutants in the cold. (A) Five-fold serial dilutions of log phase cultures were stamped onto rich medium and incubated at the indicated temperature. (B) rif1+ deletion suppresses the elongation of taz1Δ cells at 20°C. Logarithmically growing cells in rich media at 20°C are compared. (C) A mutation in the gene encoding topoisomerase II, top2-191, suppresses the severe cold sensitivity of rap1Δtaz1Δ mutants. Experiments were performed as in panel A. (D) Cell lengths in the cold. A total of 300 cells of each genotype from asynchronous cultures were analyzed. Error bars represent standard deviations.
Figure 4
Figure 4
Analysis of the effects of ectopically tethering Rap1 to telomeres. (A) Diagram of GFP-Taz1C-term and Rap1-GFP-Taz1C-term. The expressions of both chimeric constructs are driven by the nmt1 promoter. Unless otherwise indicated, all GFP-Taz1C-term experiments were performed in a taz1Δrap1+ background, and all Rap1-GFP-Taz1C-term experiments were performed in a taz1Δrap1Δ background. (B) Effects of GFP-Taz1C-term and Rap1-GFP-Taz1C-term on telomere length regulation. Experiments were performed as in Figure 1A. (C) Telomeric 3′ overhang regulation in GFP-Taz1C-term and Rap1-GFP-Taz1C-term cells. Experiments were performed as in Figure 1B. (D) Analysis of telomere fusions in GFP-Taz1C-term and Rap1-GFP-Taz1C-term cells. Experiments were performed as in Figure 2. The strain represented in lanes 1 and 2 (taz1Δrap1Δ Rap1-GFP-Taz1C-term) displays less ‘I' fragment, and correspondingly less ‘M+I' and ‘L+I' than the other strains, presumably because of mutations in the region containing the terminal Not1 site on the right arm of Chr I. (E) Growth of GFP-Taz1C-term and Rap1-GFP-Taz1C-term at 20°C. Experiments were performed as in Figure 3A.
Figure 5
Figure 5
Summary of genetic analysis of Rap1, Rif1 and Taz1. (A) Taz1-containing telomere complexes. Taz1 organizes at least three functional telomere complexes. Rif1–Taz inhibits telomere elongation. Rap1–Taz1 inhibits telomere elongation, controls 3′ overhang generation and prevents NHEJ-mediated chromosome end fusions. In addition, Taz1 functions in a Rap1/Rif1-independent manner to protect cells from cold sensitivity and to positively regulate telomere length (see text). (B) Telomere complexes lacking Taz1. In the absence of Taz1, Rap1 restrains cold-specific abnormalities while Rif1 promotes it. In taz1Δ cells, Rif1 may act at nontelomeric sites to affect telomere metabolism.

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