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Rad59-facilitated acquisition of Y' elements by short telomeres delays the onset of senescence.

Churikov D, Charifi F, Simon MN, Géli V - PLoS Genet. (2014)

Bottom Line: We found that choice of the Y' donor was not random, since both engineered telomere VII-L and native VI-R acquired Y' elements from partially overlapping sets of specific chromosome ends.Therefore, Y' translocation events taking place during presenescence are genetically separable from Rad51-dependent Y' amplification process that occurs later during type I survivor formation.We show that Rad59-facilitated Y' translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

View Article: PubMed Central - PubMed

Affiliation: Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Aix Marseille University, Institut Paoli-Calmettes, LNCC (Equipe labellisée), Marseille, France.

ABSTRACT
Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1-3-like tracts present between subtelomeric X and Y' elements, which is followed by BIR-mediated non-reciprocal translocation of Y' element and terminal TG1-3 repeats from the donor end onto the shortened telomere. We found that choice of the Y' donor was not random, since both engineered telomere VII-L and native VI-R acquired Y' elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y' translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1-3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y' translocation events taking place during presenescence are genetically separable from Rad51-dependent Y' amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y' translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

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Y′ translocation depends on Rad52 and Pol32, is facilitated by Rad59, but is largely independent of Rad51.(A) Schematic of the experiment. (B and C) The semi-quantitative junction PCR assays for Y′ element translocation on VII-L end. Tet-off TLC1 strains with indicated gene deletions were grown in the presence of Dox to suppress TLC1 expression. Abrupt shortening of the TelVII-L was induced via transient induction of Cre by shifting cells to galactose for 36 h. At the end of Cre induction, single cells were micromanipulated onto a grid of YPD agar supplemented with Dox, and the formation of microcolonies was monitored microscopically. After four days of colonies outgrowth (Figure S5), DNA was extracted from clonal populations recovered from transiently arrested cells and from bulk populations grown in liquid culture for 48 h after Cre induction. PCR was performed with the primers designed to amplify the VII-L/Y′ junction (Figure 3A). TC, template control (PCR at the very terminus of VII-L). The bracket indicates two pol32Δ clones that lost the most terminal VII-L primer site due to VII-L end rearrangement that is distinct from Y′ translocation (see Figure S6). (D) Real-time quantitative PCR analysis of the genetic requirements of Y′ translocation. The fractions of “repaired” VII-L for each genotype were calculated as follows: fraction repaired  = 2−ΔCt, ΔCt = Ct7L-Y′−Ct7L, where 7L and 7L-Y′ represent amplification of the total and “repaired” VII-L as indicated in the schematic. Data are presented as mean ±SE for 15, 6, 18, 18, and 6 random clones of WT, rad52Δ, rad51Δ, rad59Δ, and pol32Δ, respectively, isolated at ∼16 PD after Cre induction. The numbers below the graph indicate the efficiencies of VII-L repair by Y′ translocation for each genotype relative to WT (set to 100%).
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pgen-1004736-g005: Y′ translocation depends on Rad52 and Pol32, is facilitated by Rad59, but is largely independent of Rad51.(A) Schematic of the experiment. (B and C) The semi-quantitative junction PCR assays for Y′ element translocation on VII-L end. Tet-off TLC1 strains with indicated gene deletions were grown in the presence of Dox to suppress TLC1 expression. Abrupt shortening of the TelVII-L was induced via transient induction of Cre by shifting cells to galactose for 36 h. At the end of Cre induction, single cells were micromanipulated onto a grid of YPD agar supplemented with Dox, and the formation of microcolonies was monitored microscopically. After four days of colonies outgrowth (Figure S5), DNA was extracted from clonal populations recovered from transiently arrested cells and from bulk populations grown in liquid culture for 48 h after Cre induction. PCR was performed with the primers designed to amplify the VII-L/Y′ junction (Figure 3A). TC, template control (PCR at the very terminus of VII-L). The bracket indicates two pol32Δ clones that lost the most terminal VII-L primer site due to VII-L end rearrangement that is distinct from Y′ translocation (see Figure S6). (D) Real-time quantitative PCR analysis of the genetic requirements of Y′ translocation. The fractions of “repaired” VII-L for each genotype were calculated as follows: fraction repaired  = 2−ΔCt, ΔCt = Ct7L-Y′−Ct7L, where 7L and 7L-Y′ represent amplification of the total and “repaired” VII-L as indicated in the schematic. Data are presented as mean ±SE for 15, 6, 18, 18, and 6 random clones of WT, rad52Δ, rad51Δ, rad59Δ, and pol32Δ, respectively, isolated at ∼16 PD after Cre induction. The numbers below the graph indicate the efficiencies of VII-L repair by Y′ translocation for each genotype relative to WT (set to 100%).

Mentions: To get insight into the mechanism of short telomere repair, we examined its genetic requirements. To this end, we generated a set of isogenic mutants by deleting RAD52, RAD51, and RAD59 genes in a strain with the VII-L telomere modified for inducible shortening and TLC1 allele with a tetracycline-regulatable promoter (tetO2-TLC1). Upon addition of doxycycline (Dox), expression of TLC1 is tightly repressed and telomeres shorten progressively with each generation ([26] and Figure S4). We first examined the effect of gene deletions on the efficiency of Y′ translocation by semi-quantitative PCR assay. Telomerase was inactivated by addition of Dox and abrupt shortening of the VII-L was then induced following the scheme in Figure 5A. We analyzed junction PCR products in clonal populations of HR-proficient or mutant cells (Figure 5B,C and Figure S5). As expected, robust junction PCR products indicating Y′ translocation events were detected in the clonal populations of HR-proficient cells (Figure 5B). Weaker product was also detectable in the mixed population of cells grown in liquid culture (Bulk). RAD52 deletion nearly completely abolished Y′ translocation as judged by severe reduction of the amplified VII-L/Y′ junctions in either mixed or clonal populations (Figure 5B). Thus, Rad52 is essential for recombination between TG1–3 repeats that leads to Y′ translocation. Surprisingly, deletion of the RAD51 had little effect on the efficiency of Y′ translocation (Figure 5C, top panel), demonstrating that Rad51 may not be essential for recombination between TG1–3 repeats. In contrast, RAD59 deletion reduced both the amount and the heterogeneity of amplified VII-L/Y′ junctions (Figure 5C, midpanel) suggesting lower frequency of TG1–3 repeat recombination in the absence of Rad59. These results indicate that Rad51 filament assembly may not be required at the 3′ overhang of the short telomere, which pairing with an internal tract of the TG1–3 repeats could depend only on Rad52 strand annealing activity which is stimulated by Rad59.


Rad59-facilitated acquisition of Y' elements by short telomeres delays the onset of senescence.

Churikov D, Charifi F, Simon MN, Géli V - PLoS Genet. (2014)

Y′ translocation depends on Rad52 and Pol32, is facilitated by Rad59, but is largely independent of Rad51.(A) Schematic of the experiment. (B and C) The semi-quantitative junction PCR assays for Y′ element translocation on VII-L end. Tet-off TLC1 strains with indicated gene deletions were grown in the presence of Dox to suppress TLC1 expression. Abrupt shortening of the TelVII-L was induced via transient induction of Cre by shifting cells to galactose for 36 h. At the end of Cre induction, single cells were micromanipulated onto a grid of YPD agar supplemented with Dox, and the formation of microcolonies was monitored microscopically. After four days of colonies outgrowth (Figure S5), DNA was extracted from clonal populations recovered from transiently arrested cells and from bulk populations grown in liquid culture for 48 h after Cre induction. PCR was performed with the primers designed to amplify the VII-L/Y′ junction (Figure 3A). TC, template control (PCR at the very terminus of VII-L). The bracket indicates two pol32Δ clones that lost the most terminal VII-L primer site due to VII-L end rearrangement that is distinct from Y′ translocation (see Figure S6). (D) Real-time quantitative PCR analysis of the genetic requirements of Y′ translocation. The fractions of “repaired” VII-L for each genotype were calculated as follows: fraction repaired  = 2−ΔCt, ΔCt = Ct7L-Y′−Ct7L, where 7L and 7L-Y′ represent amplification of the total and “repaired” VII-L as indicated in the schematic. Data are presented as mean ±SE for 15, 6, 18, 18, and 6 random clones of WT, rad52Δ, rad51Δ, rad59Δ, and pol32Δ, respectively, isolated at ∼16 PD after Cre induction. The numbers below the graph indicate the efficiencies of VII-L repair by Y′ translocation for each genotype relative to WT (set to 100%).
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pgen-1004736-g005: Y′ translocation depends on Rad52 and Pol32, is facilitated by Rad59, but is largely independent of Rad51.(A) Schematic of the experiment. (B and C) The semi-quantitative junction PCR assays for Y′ element translocation on VII-L end. Tet-off TLC1 strains with indicated gene deletions were grown in the presence of Dox to suppress TLC1 expression. Abrupt shortening of the TelVII-L was induced via transient induction of Cre by shifting cells to galactose for 36 h. At the end of Cre induction, single cells were micromanipulated onto a grid of YPD agar supplemented with Dox, and the formation of microcolonies was monitored microscopically. After four days of colonies outgrowth (Figure S5), DNA was extracted from clonal populations recovered from transiently arrested cells and from bulk populations grown in liquid culture for 48 h after Cre induction. PCR was performed with the primers designed to amplify the VII-L/Y′ junction (Figure 3A). TC, template control (PCR at the very terminus of VII-L). The bracket indicates two pol32Δ clones that lost the most terminal VII-L primer site due to VII-L end rearrangement that is distinct from Y′ translocation (see Figure S6). (D) Real-time quantitative PCR analysis of the genetic requirements of Y′ translocation. The fractions of “repaired” VII-L for each genotype were calculated as follows: fraction repaired  = 2−ΔCt, ΔCt = Ct7L-Y′−Ct7L, where 7L and 7L-Y′ represent amplification of the total and “repaired” VII-L as indicated in the schematic. Data are presented as mean ±SE for 15, 6, 18, 18, and 6 random clones of WT, rad52Δ, rad51Δ, rad59Δ, and pol32Δ, respectively, isolated at ∼16 PD after Cre induction. The numbers below the graph indicate the efficiencies of VII-L repair by Y′ translocation for each genotype relative to WT (set to 100%).
Mentions: To get insight into the mechanism of short telomere repair, we examined its genetic requirements. To this end, we generated a set of isogenic mutants by deleting RAD52, RAD51, and RAD59 genes in a strain with the VII-L telomere modified for inducible shortening and TLC1 allele with a tetracycline-regulatable promoter (tetO2-TLC1). Upon addition of doxycycline (Dox), expression of TLC1 is tightly repressed and telomeres shorten progressively with each generation ([26] and Figure S4). We first examined the effect of gene deletions on the efficiency of Y′ translocation by semi-quantitative PCR assay. Telomerase was inactivated by addition of Dox and abrupt shortening of the VII-L was then induced following the scheme in Figure 5A. We analyzed junction PCR products in clonal populations of HR-proficient or mutant cells (Figure 5B,C and Figure S5). As expected, robust junction PCR products indicating Y′ translocation events were detected in the clonal populations of HR-proficient cells (Figure 5B). Weaker product was also detectable in the mixed population of cells grown in liquid culture (Bulk). RAD52 deletion nearly completely abolished Y′ translocation as judged by severe reduction of the amplified VII-L/Y′ junctions in either mixed or clonal populations (Figure 5B). Thus, Rad52 is essential for recombination between TG1–3 repeats that leads to Y′ translocation. Surprisingly, deletion of the RAD51 had little effect on the efficiency of Y′ translocation (Figure 5C, top panel), demonstrating that Rad51 may not be essential for recombination between TG1–3 repeats. In contrast, RAD59 deletion reduced both the amount and the heterogeneity of amplified VII-L/Y′ junctions (Figure 5C, midpanel) suggesting lower frequency of TG1–3 repeat recombination in the absence of Rad59. These results indicate that Rad51 filament assembly may not be required at the 3′ overhang of the short telomere, which pairing with an internal tract of the TG1–3 repeats could depend only on Rad52 strand annealing activity which is stimulated by Rad59.

Bottom Line: We found that choice of the Y' donor was not random, since both engineered telomere VII-L and native VI-R acquired Y' elements from partially overlapping sets of specific chromosome ends.Therefore, Y' translocation events taking place during presenescence are genetically separable from Rad51-dependent Y' amplification process that occurs later during type I survivor formation.We show that Rad59-facilitated Y' translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

View Article: PubMed Central - PubMed

Affiliation: Marseille Cancer Research Center (CRCM), U1068 Inserm, UMR7258 CNRS, Aix Marseille University, Institut Paoli-Calmettes, LNCC (Equipe labellisée), Marseille, France.

ABSTRACT
Telomerase-negative yeasts survive via one of the two Rad52-dependent recombination pathways, which have distinct genetic requirements. Although the telomere pattern of type I and type II survivors is well characterized, the mechanistic details of short telomere rearrangement into highly evolved pattern observed in survivors are still missing. Here, we analyze immediate events taking place at the abruptly shortened VII-L and native telomeres. We show that short telomeres engage in pairing with internal Rap1-bound TG1-3-like tracts present between subtelomeric X and Y' elements, which is followed by BIR-mediated non-reciprocal translocation of Y' element and terminal TG1-3 repeats from the donor end onto the shortened telomere. We found that choice of the Y' donor was not random, since both engineered telomere VII-L and native VI-R acquired Y' elements from partially overlapping sets of specific chromosome ends. Although short telomere repair was associated with transient delay in cell divisions, Y' translocation on native telomeres did not require Mec1-dependent checkpoint. Furthermore, the homeologous pairing between the terminal TG1-3 repeats at VII-L and internal repeats on other chromosome ends was largely independent of Rad51, but instead it was facilitated by Rad59 that stimulates Rad52 strand annealing activity. Therefore, Y' translocation events taking place during presenescence are genetically separable from Rad51-dependent Y' amplification process that occurs later during type I survivor formation. We show that Rad59-facilitated Y' translocations on X-only telomeres delay the onset of senescence while preparing ground for type I survivor formation.

Show MeSH