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

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 null 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

El mecanisme de vigilància de la fase S estabilitza els nivells de la ciclina Clb6 en resposta a estrès genotòxic

Les cèl·lules eucariotes estan constantment exposades a dany al DNA i a estrès replicatiu (estrès genotòxic), font d'inestabilitat genòmica. L'anomenat mecanisme de vigilància (checkpoint) de la fase S detecta la presència d'estrès genotòxic i genera una resposta que inclou l'aturada preventiva de la replicació i del cicle cel·lular, per tal de donar temps a la superació del problema, i la protecció de la replicació dels cromosomes. Quan els elements centrals d'aquest mecanisme de control estan mutants, les cèl·lules esdevenen genèticament inestables i, en humans, predisposa al càncer. Malgrat el seu paper crític en la preservació de la integritat del genoma, es desconeix la manera com el mecanisme de vigilància de la fase S executa bona part de la seva resposta. Els elements i vies que constitueixen el checkpoint estan conservats evolutivament. El nostre treball s'ha centrat en el llevat de gemmació Saccharomyces cerevisiae. En aquest organisme la proteïna quinasa efectora principal és Rad53. Amb l'objectiu de descobrir dianes a través de les quals Rad53 regula la fase S, s'han assajat en una reacció quinasa in vitro diferents proteïnes candidates a ser substrat de Rad53. Això ha permès la identificació de la ciclina de fase S Clb6 com a substrat de Rad53 in vitro.Les ciclines són les subunitats activadores de la quinasa dependent de ciclina Cdc28/Cdk1, que promou la progressió del cicle cel·lular. Cdc28/Cdk1 és activada per diferents ciclines específiques de les diferents fases del cicle cel·lular, que li confereixen especificitat de substrat. Clb6 és una de les dues ciclines de fase S (junt amb Clb5), que promouen la replicació del DNA. Mentre que Clb5 és present des del final de la fase G1 fins a anafase, Clb6 és eliminada durant la primera meitat de la fase S. Per tal d'estudiar si Clb6 és diana de Rad53 in vivo, s'ha estudiat el seu comportament en resposta a estrès genotòxic. Interessantment, en una fase S compromesa els nivells de Clb6 es mantenen estables, i aquesta estabilització és dependent de Rad53. Experiments amb l'inhibidor de la traducció proteica cicloheximida mostren que l'estabilització de Clb6 requereix síntesi de novo. Donat que en un cicle cel·lular no pertorbat Clb6 és expressada exclusivament en fase G1, sota el control del factor de transcripció MBF. Els nostres resultats indiquen que MBF es reactiva en una fase S compromesa: la subunitat transactivadora Swi6, exclosa del nucli en una fase S no pertorbada, és nuclear en una fase S compromesa; per altra banda, la sobreexpressió d'una forma hiperestable del repressor d'MBF Nrm1 dóna lloc a la desaparició de Clb6 malgrat la presència continuada d'estrès genotòxic i una resposta checkpoint funcional. Fins ara MBF era considerat un factor de transcripció exclusiu de la fase G1.Per últim hem explorat el paper de l'estabilització de Clb6 en una fase S compromesa. El mutant nul clb6 no presenta cap fenotip detectable, suggerint que Clb6 pot operar en forma redundant dins de la resposta a estrès genotòxic. Per aquest motiu hem optat pel plantejament complementari: estudiar l'efecte de mantenir la presència de Clb6 en una fase S no pertorbada (i per tant en absència de la resta de la resposta checkpoint). En aquestes condicions les cèl·lules repliquen el seu DNA amb aparent normalitat, però queden aturades en un estadi previ a anafase. Aquests resultats suggereixen que Clb6 pot ser un element efector de la branca S-M del mecanisme de vigilància de la fase S, que estaria constituïda per diversos punts de control solapat. Aquest paper justificaria que Clb6 hagi de ser eliminada de forma avançada respecte a Clb5.Eukaryotic cells are permanently exposed to DNA damage and to replication stress (genotoxic stress), a source of genomic instability. A surveillance mechanism, the S phase checkpoint, detects and responds to genotoxic stress, and elicits a response that includes the arrest of cell cycle progression -to give time to overcome the insult- and the protection of chromosome replication. When the checkpoint central elements are mutated cells become genetically unstable, which in humans results in a high frequency of cancer.Despite such critical role to preserve genomic integrity, much remains unknown about the way how the S phase checkpoint executes its response. Because the checkpoint elements and pathways are conserved, our work has been carried out in the budding yeast Saccharomyces cerevisiae. In this organism Rad53 is the S phase checkpoint effector kinase. To identify novel Rad53 targets, we carried out an in vitro kinase assay with different potential substrate proteins involved in cell cycle control. This approach identified the S phase cyclin Clb6 as an in vitro Rad53 substrate.Cyclins are the activatory subunits for Cdc28/Cdk1, the cyclin dependent kinase that drives cell cycle progression in budding yeast. Cdc28/Cdk1 is activated by different cell cycle phase specific cyclins, that also confer substrate specificity to the kinase. Clb6 and Clb5 are the S phase cyclins, promoting the onset of DNA replication. Interestingly, whereas the presence of Clb5 spans from late G1 to anaphase, Clb6 is eliminated earlier, during S phase.To study whether Clb6 is a target of Rad53 in vivo, we explored the effect of genotoxic stress on the cyclin. When cells are exposed to replication stress or to DNA damage during S phase, the presence of Clb6 is stabilized, and such stabilization depends on Rad53.Experiments with the inhibitor of protein translation cycloheximide show that the stabilization of Clb6 requires de novo synthesis. In an unperturbed cell cycle Clb6 is expressed only during G1 phase, under the control of the transcription factor MBF. Our results show that MBF is reactivated in a compromised S phase: the transactivatory subunit Swi6, excluded from the nucleus in an unperturbed S phase, shows nuclear localization during a compromised S phase; in addition, the overexpression of a hyperstable form of the MBF repressor Nrm1 results in Clb6 elimination, despite the continued presence of genotoxic stress and a functional checkpoint response. To date, MBF was considered a transcription factor operating solely and characteristically during G1 phase.Finally we have explored the role of Clb6 stabilization in response to genotoxic stress. The clb6 null mutant does not present a detectable phenotype, which is compatible with a redundant role in the response to genotoxic stress. We therefore chose to address the question the other way round, and study the effect of keeping the levels of Clb6 stable during an otherwise unperturbed S phase (and therefore in the absence of the vast checkpoint response that could mask the specific role of Clb6). Under such conditions cells replicate normally, but arrest in a stage previous to anaphase. These results suggest that Clb6 may be an effector element in the S-M branch of the S phase checkpoint. One such role would explain why Clb6 must be eliminated in S phase, ahead of Clb5 during mitosis

Adaptación y conservación de los aparatos de ortodoncia mediante aula invertida en el módulo de Prótesis y ortodoncia de Técnico Superior en Higiene Bucodental

The following final master's thesis presents an intervention proposal for the studies of Oral Hygiene. The main objective is to implement the Flipped Classroom methodology in the Prosthetics and Orthodontics module to develop the contents of the unit of "adaptation and conservation of orthodontic appliances". In the first part, a bibliographic search is carried out to describe the evolution of vocational training in Spain. It details the emergence of new teaching methodologies, as well as the incorporation of ICT in the sector. After that, the Flipped Classroom methodology is described; as its phases and its use in the teaching of the health field. In the second part, the unit of “adaptation and conservation of the orthodontic appliance” is developed, applying the Flipped Classroom methodology during the different sessions and activities. It is concluded that the use of the Flipped Classroom aims to motivate students and encourage deep learning, so that they can apply the knowledge in their future work.En el siguiente trabajo final de máster se presenta una propuesta de intervención en el Ciclo Formativo de Grado Superior (CFGS) de Higiene Bucodental. El objetivo principal es implementar la metodología del Aula Invertida en el módulo de Prótesis y Ortodoncia para desarrollar los contenidos de la unidad de trabajo de “adaptación y conservación de los aparatos de ortodoncia”. En una primera parte se realiza una búsqueda bibliográfica para describir la evolución de la formación profesional en España hasta la actualidad. Se detalla la aparición de las nuevas metodologías didácticas, así como la incorporación de las tecnologías de la información y comunicación (TIC) en el sector. Seguidamente, se describe la metodología del aula invertida, sus fases y su uso en la docencia del ámbito sanitario. En una segunda parte, se desarrolla la unidad de trabajo de “adaptación y conservación del aparato de ortodoncia” aplicando la metodología del aula invertida durante las distintas sesiones y actividades. Se concluye que el uso del aula invertida pretende motivar a los alumnos y fomentar el aprendizaje profundo, de manera que puedan aplicar los conocimientos en su futuro laboral

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

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 null 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

Three Different Pathways Prevent Chromosome Segregation in the Presence of DNA Damage or Replication Stress in Budding Yeast

<div><p>A surveillance mechanism, the S phase checkpoint, blocks progression into mitosis in response to DNA damage and replication stress. Segregation of damaged or incompletely replicated chromosomes results in genomic instability. In humans, the S phase checkpoint has been shown to constitute an anti-cancer barrier. Inhibition of mitotic cyclin dependent kinase (M-CDK) activity by Wee1 kinases is critical to block mitosis in some organisms. However, such mechanism is dispensable in the response to genotoxic stress in the model eukaryotic organism <i>Saccharomyces cerevisiae</i>. We show here that the Wee1 ortholog Swe1 does indeed inhibit M-CDK activity and chromosome segregation in response to genotoxic insults. Swe1 dispensability in budding yeast is the result of a redundant control of M-CDK activity by the checkpoint kinase Rad53. In addition, our results indicate that Swe1 is an effector of the checkpoint central kinase Mec1. When checkpoint control on M-CDK and on Pds1/securin stabilization are abrogated, cells undergo aberrant chromosome segregation.</p></div

G1 cyclin driven DNA replication

<p>The mitotic cell cycle is driven by Cyclin-Dependent Kinases (CDK). CDK activation requires the binding of activatory subunits termed cyclins. Different waves of cyclins are expressed during the cell cycle, enabling CDKs to trigger phase specific events. For instance, S phase cyclins promote the initiation of DNA replication but not chromosome segregation. There are at least 2 explanations for how such regulation is achieved. According to one of the visions, cyclins confer intrinsic substrate specificity to the CDK catalytic subunit. Alternatively a quantitative model has been proposed, according to which ever-increasing CDK activity is required to trigger cell cycle events from G1 to M. If a quantitative control prevails, then an early cyclin should trigger later cycle events if accumulated at high enough levels at the right time and place. We show here that a G1 phase cyclin bears the potential to trigger DNA replication and promote S and G2 phase specific transcription.</p

Three different pathways prevent chromosome segregation in the presence of genotoxic stress.

<p>Molecular diagram showing the three pathways that block Mitotic Cyclin Dependent Kinase activity and anaphase in response to genotoxic stress. Swe1 and the S phase checkpoint effector kinase Rad53 are individually sufficient to block M-CDK activity. Our results place Swe1 as a downstream effector of the S phase checkpoint. M-CDK inhibition and Pds1/securin stabilization are individually sufficient to prevent anaphase. Only when the three pathways are disrupted cells fail to block the segregation of damaged or incompletely replicated chromosomes. In grey, regulatory pathways taken from previous works [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref023" target="_blank">23</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref028" target="_blank">28</a>]. The Spindle Assembly Checkpoint (SAC) is included in the model as it may also play a role in Pds1 stabilization [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005468#pgen.1005468.ref076" target="_blank">76</a>].</p

Neither unrestrained M-CDK activity alone nor lack of Pds1/securin alone are enough to abrogate the block of chromosome segregation in the presence of DNA damage.

<p>(A) Mutant <i>rad53 swe1</i> cells remain competent to block the segregation of damaged chromosomes. Percentage of cells showing segregated masses of DNA. Wild type (WT, strain YGP20), <i>rad53 swe1</i> (strain YGP121), <i>rad53</i> (strain YGP38), and <i>swe1</i> (strain YGP98) cells were grown to mid-exponential phase (Log), synchronized in G1 phase with the pheromone alpha-factor (G1), then released into S phase in the presence of 0.033% MMS. Cells were collected at the indicated times (min). Fixed cells were stained with DAPI to visualize DNA by fluorescence microscopy. 120 cells were counted in each of 3 independent experiments. Data are represented as mean ± SD (error bars). (B) Representative cells 240 minutes after the release from G1. (C-D) The absence of Pds1/securin is not sufficient to allow chromosome segregation in the presence of DNA damage in S phase. Wild type cells (WT, YGP20) and null <i>pds1</i> cells (strain YRP33) were treated and analyzed as in (A-B).</p