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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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Clones derived from transiently arrested cells exhibit VII-L end rearrangement consistent with Y′ element translocation.(A) Microcolony formation assay was performed to identify cells undergoing transient arrest after telomerase inactivation. Single cells from two double Cre-loxP strains that differ in a length of TelVII-L were micromanipulated onto a grid on YPD agar plate at 36 h (∼15 PD) after induction of Cre expression in liquid culture. Cell divisions were monitored microscopically and the numbers of cells in microcolonies were counted at 4 and 6 h after plating. Representative images of the plates taken after 3 days of colonies outgrowth are shown. The positions where cell division arrest was detected at the time of plating are circled. (B) Histogram showing the fraction of cells which arrested divisions either during the first or second cell cycle after plating. Mean values ±SE for two independent micromanipulations at 15 and 18 PD after Cre induction are shown. One-sided chi-square test was used to evaluate the significance of the difference in overall fraction of arrested cells. χ2 (1, N = 162)  = 3.08, p = 0.040. (C) Southern blot analysis of the VII-L end in the clones derived from cells recovered from transient arrest (A). The lanes marked with white and grey boxes correspond to “no delay” control and “growth-delayed” clones, respectively. The colonies were inoculated in 3 ml of YPD and cultured overnight to generate sufficient number of cells for DNA extraction. For each clone two aliquots of DNA were digested separately with PacI and MfeI, which recognition site positions at the VII-L end are shown in the diagram. Digested DNA was subjected to Southern blot analyses with VII-L-specific probe (black rectangle in the scheme). The brackets indicate terminal fragments of the TelVII-L, whereas open arrowheads point to the high molecular weight fragments resulted from VII-L end rearrangement.
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pgen-1004736-g002: Clones derived from transiently arrested cells exhibit VII-L end rearrangement consistent with Y′ element translocation.(A) Microcolony formation assay was performed to identify cells undergoing transient arrest after telomerase inactivation. Single cells from two double Cre-loxP strains that differ in a length of TelVII-L were micromanipulated onto a grid on YPD agar plate at 36 h (∼15 PD) after induction of Cre expression in liquid culture. Cell divisions were monitored microscopically and the numbers of cells in microcolonies were counted at 4 and 6 h after plating. Representative images of the plates taken after 3 days of colonies outgrowth are shown. The positions where cell division arrest was detected at the time of plating are circled. (B) Histogram showing the fraction of cells which arrested divisions either during the first or second cell cycle after plating. Mean values ±SE for two independent micromanipulations at 15 and 18 PD after Cre induction are shown. One-sided chi-square test was used to evaluate the significance of the difference in overall fraction of arrested cells. χ2 (1, N = 162)  = 3.08, p = 0.040. (C) Southern blot analysis of the VII-L end in the clones derived from cells recovered from transient arrest (A). The lanes marked with white and grey boxes correspond to “no delay” control and “growth-delayed” clones, respectively. The colonies were inoculated in 3 ml of YPD and cultured overnight to generate sufficient number of cells for DNA extraction. For each clone two aliquots of DNA were digested separately with PacI and MfeI, which recognition site positions at the VII-L end are shown in the diagram. Digested DNA was subjected to Southern blot analyses with VII-L-specific probe (black rectangle in the scheme). The brackets indicate terminal fragments of the TelVII-L, whereas open arrowheads point to the high molecular weight fragments resulted from VII-L end rearrangement.

Mentions: To isolate the cells undergoing cell cycle arrest due to TG1–3 tract shortening beyond the 60 bp threshold, we conducted clonal analysis of the telomerase-negative cultures at ∼15 PD after Cre induction. To this end, single cells were micromanipulated on a grid of agar, and analyzed for their ability to form microcolonies. While many cells divided regularly, at least once every 2 hours, and formed microcolonies of more than 16 cells after 8 hours on agar, a fraction of cells never divided during this time or stopped dividing at the 2- or 4-cell stage. These arrested microcolonies were marked (Figure 2A). Unexpectedly, most of the cells, which initially failed to divide, formed colonies after four days at 30°C (Figure 2A). Therefore, the majority of cells was able to overcome cell cycle arrest and resumed divisions. The fraction of arrested cells was significantly greater in the strain with shortened TelVII-L compared to the control strain (Figure 2B and Figure S2) indicating that shortening of a single telomere aggravated the effect of telomerase inactivation on cell cycle progression.


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)

Clones derived from transiently arrested cells exhibit VII-L end rearrangement consistent with Y′ element translocation.(A) Microcolony formation assay was performed to identify cells undergoing transient arrest after telomerase inactivation. Single cells from two double Cre-loxP strains that differ in a length of TelVII-L were micromanipulated onto a grid on YPD agar plate at 36 h (∼15 PD) after induction of Cre expression in liquid culture. Cell divisions were monitored microscopically and the numbers of cells in microcolonies were counted at 4 and 6 h after plating. Representative images of the plates taken after 3 days of colonies outgrowth are shown. The positions where cell division arrest was detected at the time of plating are circled. (B) Histogram showing the fraction of cells which arrested divisions either during the first or second cell cycle after plating. Mean values ±SE for two independent micromanipulations at 15 and 18 PD after Cre induction are shown. One-sided chi-square test was used to evaluate the significance of the difference in overall fraction of arrested cells. χ2 (1, N = 162)  = 3.08, p = 0.040. (C) Southern blot analysis of the VII-L end in the clones derived from cells recovered from transient arrest (A). The lanes marked with white and grey boxes correspond to “no delay” control and “growth-delayed” clones, respectively. The colonies were inoculated in 3 ml of YPD and cultured overnight to generate sufficient number of cells for DNA extraction. For each clone two aliquots of DNA were digested separately with PacI and MfeI, which recognition site positions at the VII-L end are shown in the diagram. Digested DNA was subjected to Southern blot analyses with VII-L-specific probe (black rectangle in the scheme). The brackets indicate terminal fragments of the TelVII-L, whereas open arrowheads point to the high molecular weight fragments resulted from VII-L end rearrangement.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222662&req=5

pgen-1004736-g002: Clones derived from transiently arrested cells exhibit VII-L end rearrangement consistent with Y′ element translocation.(A) Microcolony formation assay was performed to identify cells undergoing transient arrest after telomerase inactivation. Single cells from two double Cre-loxP strains that differ in a length of TelVII-L were micromanipulated onto a grid on YPD agar plate at 36 h (∼15 PD) after induction of Cre expression in liquid culture. Cell divisions were monitored microscopically and the numbers of cells in microcolonies were counted at 4 and 6 h after plating. Representative images of the plates taken after 3 days of colonies outgrowth are shown. The positions where cell division arrest was detected at the time of plating are circled. (B) Histogram showing the fraction of cells which arrested divisions either during the first or second cell cycle after plating. Mean values ±SE for two independent micromanipulations at 15 and 18 PD after Cre induction are shown. One-sided chi-square test was used to evaluate the significance of the difference in overall fraction of arrested cells. χ2 (1, N = 162)  = 3.08, p = 0.040. (C) Southern blot analysis of the VII-L end in the clones derived from cells recovered from transient arrest (A). The lanes marked with white and grey boxes correspond to “no delay” control and “growth-delayed” clones, respectively. The colonies were inoculated in 3 ml of YPD and cultured overnight to generate sufficient number of cells for DNA extraction. For each clone two aliquots of DNA were digested separately with PacI and MfeI, which recognition site positions at the VII-L end are shown in the diagram. Digested DNA was subjected to Southern blot analyses with VII-L-specific probe (black rectangle in the scheme). The brackets indicate terminal fragments of the TelVII-L, whereas open arrowheads point to the high molecular weight fragments resulted from VII-L end rearrangement.
Mentions: To isolate the cells undergoing cell cycle arrest due to TG1–3 tract shortening beyond the 60 bp threshold, we conducted clonal analysis of the telomerase-negative cultures at ∼15 PD after Cre induction. To this end, single cells were micromanipulated on a grid of agar, and analyzed for their ability to form microcolonies. While many cells divided regularly, at least once every 2 hours, and formed microcolonies of more than 16 cells after 8 hours on agar, a fraction of cells never divided during this time or stopped dividing at the 2- or 4-cell stage. These arrested microcolonies were marked (Figure 2A). Unexpectedly, most of the cells, which initially failed to divide, formed colonies after four days at 30°C (Figure 2A). Therefore, the majority of cells was able to overcome cell cycle arrest and resumed divisions. The fraction of arrested cells was significantly greater in the strain with shortened TelVII-L compared to the control strain (Figure 2B and Figure S2) indicating that shortening of a single telomere aggravated the effect of telomerase inactivation on cell cycle progression.

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