Inhibition of the mitotic exit network in response to damaged telomeres

Abstract

When chromosomal DNA is damaged, progression through the cell cycle is halted to provide the cells with time to repair the genetic material before it is distributed between the mother and daughter cells. In Saccharomyces cerevisiae, this cell cycle arrest occurs at the G2/M transition. However, it is also necessary to restrain exit from mitosis by maintaining Bfa1-Bub2, the inhibitor of the Mitotic Exit Network (MEN), in an active state. While the role of Bfa1 and Bub2 in the inhibition of mitotic exit when the spindle is not properly aligned and the spindle position checkpoint is activated has been extensively studied, the mechanism by which these proteins prevent MEN function after DNA damage is still unclear. Here, we propose that the inhibition of the MEN is specifically required when telomeres are damaged but it is not necessary to face all types of chromosomal DNA damage, which is in agreement with previous data in mammals suggesting the existence of a putative telomere-specific DNA damage response that inhibits mitotic exit. Furthermore, we demonstrate that the mechanism of MEN inhibition when telomeres are damaged relies on the Rad53-dependent inhibition of Bfa1 phosphorylation by the Polo-like kinase Cdc5, establishing a new key role of this kinase in regulating cell cycle progression.

Figure 1. Inhibition of mitotic exit is specifically required after DNA damage to telomeres.

(A–B) cdc13-1 (F965), cdc13-1 rad53Δ sml1Δ (F1662), cdc13-1 bfa1Δ (F1023), cdc13-1 bub2Δ (F1787) and cdc13-1 bfa1Δ bub2Δ (F1786) cells were grown in YPD at 23°C and arrested in G1 with pheromone. Cells were then released into YPD at 34°C and the percentages of large budded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (C) Wild type (F1587), rad53Δ sml1Δ (F1019) and bfa1Δ (F1589) cells were grown in YPD at 25°C and arrested in G1 with pheromone. After pheromone washout, cells were released into YPD containing zeocin (50 µg/µl) and the percentages of large budded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (D) rad53Δ sml1Δ (F1679), bfa1Δ (F1606), and otherwise wild type cells (F1496) carrying an HO recognition site in chromosome II and the HO endonuclease under the control of a galactose-inducible promoter were grown at 25°C in rich media with 2% raffinose and synchronized in G1 using pheromone. After release from the G1-arrest, cells were grown at 25°C in rich media with 4% galactose to induce HO expression. The percentages of large budded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (E–G) Wild type (F1587), rad53Δ sml1Δ (F1019), bfa1Δ (F1589) and rad53Δ sml1Δ bfa1Δ (F1661) cells were plated by spotting 10-fold serial dilutions of a liquid culture (OD₆₀₀ = 0.3) on YPD (E–F) or minimal media (G) plates and then incubated at 30°C. (E) Before being plated, cells were irradiated with γ-rays (300 Gy) or UV (20 Jul/m²). (F–G) The cells were plated in media containing camptothecin (7.5 µg/µl), MMS (0.015%) or HU (100 mM), as indicated.

Figure 2. Bfa1 is hypo-phosphorylated by the DDC in a Rad53-dependent manner.

(A–F) cdc15-2 (F1492), cdc13-1 cdc15-2 (F1488), cdc13-1 rad53-21 cdc15-2 (F1453), cdc13-1 chk1Δ cdc15-2 (F1782), and cdc13-1 rad53-21 chk1Δ cdc15-2 (F1784) cells expressing 3HA-Bfa1 were arrested in G1 with pheromone in YPD at 23°C, and then released into pheromone-free medium at 34°C. (A) Representative images of the cells (DIC), as well as the spindle (tubulin) and nuclear (DAPI) morphologies, are displayed for each strain at the final cell cycle arrest. (B, E) The percentages of metaphase and/or anaphase cells were determined for each of the strains at the indicated time points. (C,D,F) 3HA-Bfa1 phosphorylation was analyzed by Western blot at the indicated time points. An unspecific band was used as a loading control (LC).

Figure 3. Rad53 prevents Bfa1 phosphorylation by the Polo-like kinase Cdc5.

(A–C) cdc13-1 cdc15-2 (F1488), cdc13-1 rad53-21 cdc15-2 (F1453) and cdc13-1 rad53-21 cdc5-2 (F1316) cells expressing 3HA-Bfa1 were arrested in G1 with pheromone in YPD at 23°C, and then released into pheromone-free medium at 34°C. (A) The percentages of metaphase and anaphase cells were determined for each of the strains at the indicated time points. (B–C) 3HA-Bfa1 phosphorylation was analyzed by Western blot at the indicated time points. An unspecific band was used as a loading control (LC). (D–F) 3HA-Cdc5 localization in cdc15-2 (F1822), cdc13-1 cdc15-2 (F1824), and cdc13-1 rad53-21 cdc15-2 (F1825) cells. The cells were arrested in G1 at 23°C and then released at 34°C. The morphology of the cells (DIC), the spindle (tubulin) and nuclear (DAPI) morphologies, as well as a merged image (merged) are also displayed.

Figure 4. Bfa1 phosphorylation in the DDC-induced arrest is not dependent on CDK or Kin4.

(A) Cells expressing 3HA-Bfa1 and carrying the cdc13-1 allele alone (F1182) or in combination with either cdc28-as1 (F1184), cdc5-as1 (F1280) or both analogue-sensitive alleles (F1516) were arrested in G1 with pheromone in YPD at 23°C, released into pheromone-free medium containing the CMK-C1 (5 µM) and 1-NM-PP1 (500 nM) inhibitors, and incubated at 34°C. 3HA-Bfa1 phosphorylation was determined as in Figure 3C. (B) cdc13-1 (F965), cdc13-1 kin4Δ (F973) and cdc13-1 bfa1Δ (F1023) cells were grown in YPD at 23°C, arrested in G1 with pheromone, and released into YPD at 34°C. The percentages of large budded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (C) Cells expressing 3HA-Bfa1 and carrying the cdc15-as1 allele alone (F1068) or in combination with cdc13-1 (F1099) or cdc13-1 and kin4Δ (F1117) were arrested in G1 with pheromone in YPD at 23°C, released into pheromone-free medium containing the 1-NA-PP1 (10 µM) inhibitor, and incubated at 34°C. 3HA-Bfa1 phosphorylation was determined as in Figure 3C. (D–E) cdc13-1 cdc15-2 (F1488), cdc13-1 bub2Δ cdc15-2 (F1879), cdc13-1 rad53-21 cdc15-2 (F1453), and cdc13-1 rad53-21 bub2Δ cdc15-2 (F1880) cells expressing 3HA-Bfa1 were arrested in G1 with pheromone in YPD at 23°C, and then released into pheromone-free medium at 34°C. (D) 3HA-Bfa1 phosphorylation was analyzed by Western blot at the indicated time points. An unspecific band was used as a loading control (LC). (E) The percentages of metaphase and anaphase cells were determined for each of the strains at the indicated time points.

Figure 5. Mutants affected in Rad53 and Bfa1 display different phenotypes.

(A–B) cdc13-1 (F965), cdc13-1 rad53-21 (F1228), cdc13-1 bfa1Δ (F1023) and cdc13-1 rad53-21 bfa1Δ (F1229) cells were grown in YPD at 23°C and arrested in G1 with pheromone. (A) Cells were then released into YPD at 34°C and the percentages of large budded and rebudded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (B) Representative images of rebudded cells (DIC) and the nuclear morphology (DAPI) are also presented. (C) Wild type (F1587), cdc13-1 (F965), cdc13-1 rad53-21 (F1228) and cdc13-1 bfa1Δ (F1023) cells were plated by spotting 10-fold serial dilutions of a liquid culture (OD₆₀₀ = 0.3) on YPD and then incubated at 23, 27 or 30°C.

Figure 6. Genetic analysis of the Rad53-Cdc5-Bfa1 pathway.

(A–B) cdc13-1 (F965), cdc13-1 rad53-21 (F1228), cdc13-1 rad53-21 cdc5-2 (F1315), cdc13-1 rad53-21 cdc5-2 bfa1Δ (F1378), cdc13-1 BFA1-4A (F1396) and cdc13-1 rad53-21 BFA1-4A (F1397) cells were grown in YPD at 23°C and arrested in G1 with pheromone. Cells were then released into YPD at 34°C and the percentages of large budded and rebudded cells were determined at the indicated time points. Error bars indicate SD (n = 3). (C) Model for Rad53-dependent cell cycle arrest in response to damaged telomeres.