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A role for the spindle assembly checkpoint in the DNA damage response

View Article: PubMed Central - PubMed

ABSTRACT

Spontaneous DNA damage poses a continuous threat to genomic integrity. If unchecked, genotoxic insults result in genomic instability, a hallmark of cancer cells. In eukaryotic cells a DNA Damage Response (DDR) detects and responds to genotoxic stress, acting as an anti-cancer barrier in humans. Among other actions, the DDR blocks the segregation of incompletely replicated or damaged chromosomes, thus preventing aneuploidy. In a work aimed at better understanding such S-M control, we recently showed that cells block anaphase through different control pathways. The S phase checkpoint kinase Mec1/ATR inhibits mitotic Cyclin Dependent Kinase activity through effector kinases Swe1/Wee1 and Rad53/Chk2. Cells also stabilize the levels of Pds1/securin to block sister chromatid segregation in response to DNA damage. We show here that Pds1/securin abundance is still secured when the S phase checkpoint response is fully abrogated in mec1/ATR tel1/ATM double mutants. When such cells are exposed to genotoxic stress, Pds1/securin is stabilized in a spindle assembly checkpoint (SAC) dependent manner. Disruption of the SAC and the S phase checkpoint together, allows chromosome segregation in the presence of DNA damage or replication stress. Our results place the SAC as a part of the DDR, which appears to count on different, independent control layers to preserve genomic integrity when chromosome replication is challenged.

No MeSH data available.


Related in: MedlinePlus

Levels of Pds1/securin in the presence of genotoxic stress in S phase checkpoint and SAC mutants. Cultures of mec1∆ tel1∆ cells (a) or mec1∆ tel1∆ mad2∆ cells (b) were grown to mid-exponential phase, synchronized in G1 phase with the pheromone alpha-factor, then released into S phase in the presence of 0.2 M HU, 0.033 % MMS, or in the absence of genotoxic stress (YPD). Cells were collected at the indicated times (min). All strains are sml1∆ isogenic, to rescue the lethality of the Mec1 deletion. As a measure of synchronicity and cell cycle progression, the upper panels show the budding indexes (BI  %) and cell densities of the cultures (average of 3 independent experiments). The lower panels show representative Pds1/securin immunoblots on whole cell extracts. A Ponceau S-stained region of the same membrane used for Western blotting is shown as a loading control
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Fig2: Levels of Pds1/securin in the presence of genotoxic stress in S phase checkpoint and SAC mutants. Cultures of mec1∆ tel1∆ cells (a) or mec1∆ tel1∆ mad2∆ cells (b) were grown to mid-exponential phase, synchronized in G1 phase with the pheromone alpha-factor, then released into S phase in the presence of 0.2 M HU, 0.033 % MMS, or in the absence of genotoxic stress (YPD). Cells were collected at the indicated times (min). All strains are sml1∆ isogenic, to rescue the lethality of the Mec1 deletion. As a measure of synchronicity and cell cycle progression, the upper panels show the budding indexes (BI  %) and cell densities of the cultures (average of 3 independent experiments). The lower panels show representative Pds1/securin immunoblots on whole cell extracts. A Ponceau S-stained region of the same membrane used for Western blotting is shown as a loading control

Mentions: Since deletion of Mec1/ATR is sufficient to abrogate the regulation of mitotic Cdk1 activity (Palou et al. 2015), we next checked the levels of Pds1/securin, the third S-M control branch. To discard a contribution from the Mec1/ATR paralog kinase Tel1/ATM, both kinases were deleted this time. As shown in Fig. 2a, the double deletion mutant tel1∆ mec1∆ is still able to keep stable levels of Pds1/securin when exposed to DNA damage during S phase (MMS) or to replication stress (hydroxyurea, HU). These results indicate that an alternative control, independent of Mec1/ATR and Tel1/ATM signaling, avoids that Pds1/securin is removed when cells are exposed to genotoxic stress.Fig. 2


A role for the spindle assembly checkpoint in the DNA damage response
Levels of Pds1/securin in the presence of genotoxic stress in S phase checkpoint and SAC mutants. Cultures of mec1∆ tel1∆ cells (a) or mec1∆ tel1∆ mad2∆ cells (b) were grown to mid-exponential phase, synchronized in G1 phase with the pheromone alpha-factor, then released into S phase in the presence of 0.2 M HU, 0.033 % MMS, or in the absence of genotoxic stress (YPD). Cells were collected at the indicated times (min). All strains are sml1∆ isogenic, to rescue the lethality of the Mec1 deletion. As a measure of synchronicity and cell cycle progression, the upper panels show the budding indexes (BI  %) and cell densities of the cultures (average of 3 independent experiments). The lower panels show representative Pds1/securin immunoblots on whole cell extracts. A Ponceau S-stained region of the same membrane used for Western blotting is shown as a loading control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC5383677&req=5

Fig2: Levels of Pds1/securin in the presence of genotoxic stress in S phase checkpoint and SAC mutants. Cultures of mec1∆ tel1∆ cells (a) or mec1∆ tel1∆ mad2∆ cells (b) were grown to mid-exponential phase, synchronized in G1 phase with the pheromone alpha-factor, then released into S phase in the presence of 0.2 M HU, 0.033 % MMS, or in the absence of genotoxic stress (YPD). Cells were collected at the indicated times (min). All strains are sml1∆ isogenic, to rescue the lethality of the Mec1 deletion. As a measure of synchronicity and cell cycle progression, the upper panels show the budding indexes (BI  %) and cell densities of the cultures (average of 3 independent experiments). The lower panels show representative Pds1/securin immunoblots on whole cell extracts. A Ponceau S-stained region of the same membrane used for Western blotting is shown as a loading control
Mentions: Since deletion of Mec1/ATR is sufficient to abrogate the regulation of mitotic Cdk1 activity (Palou et al. 2015), we next checked the levels of Pds1/securin, the third S-M control branch. To discard a contribution from the Mec1/ATR paralog kinase Tel1/ATM, both kinases were deleted this time. As shown in Fig. 2a, the double deletion mutant tel1∆ mec1∆ is still able to keep stable levels of Pds1/securin when exposed to DNA damage during S phase (MMS) or to replication stress (hydroxyurea, HU). These results indicate that an alternative control, independent of Mec1/ATR and Tel1/ATM signaling, avoids that Pds1/securin is removed when cells are exposed to genotoxic stress.Fig. 2

View Article: PubMed Central - PubMed

ABSTRACT

Spontaneous DNA damage poses a continuous threat to genomic integrity. If unchecked, genotoxic insults result in genomic instability, a hallmark of cancer cells. In eukaryotic cells a DNA Damage Response (DDR) detects and responds to genotoxic stress, acting as an anti-cancer barrier in humans. Among other actions, the DDR blocks the segregation of incompletely replicated or damaged chromosomes, thus preventing aneuploidy. In a work aimed at better understanding such S-M control, we recently showed that cells block anaphase through different control pathways. The S phase checkpoint kinase Mec1/ATR inhibits mitotic Cyclin Dependent Kinase activity through effector kinases Swe1/Wee1 and Rad53/Chk2. Cells also stabilize the levels of Pds1/securin to block sister chromatid segregation in response to DNA damage. We show here that Pds1/securin abundance is still secured when the S phase checkpoint response is fully abrogated in mec1/ATR tel1/ATM double mutants. When such cells are exposed to genotoxic stress, Pds1/securin is stabilized in a spindle assembly checkpoint (SAC) dependent manner. Disruption of the SAC and the S phase checkpoint together, allows chromosome segregation in the presence of DNA damage or replication stress. Our results place the SAC as a part of the DDR, which appears to count on different, independent control layers to preserve genomic integrity when chromosome replication is challenged.

No MeSH data available.


Related in: MedlinePlus