<p>To ensure genomic integrity, dividing cells implement multiple checkpoint pathways during the course of the cell cycle. In response to DNA damage, cells may either halt the progression of the cycle (cell cycle arrest) or undergo apoptosis. This choice depends on the extent of damage and the cell's capacity for DNA repair. Cell cycle arrest induced by double-stranded DNA breaks relies on the activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell cycle effectors (e.g., Chk2 and p53) to inhibit cell cycle progression. ATM is an S/T-Q directed kinase that is critical for the cellular response to double-stranded DNA breaks. Following DNA damage, ATM is activated and recruited to sites of DNA damage by the MRN protein complex (Mre11-Rad50-Nbs1 proteins) where ATM phosphorylates multiple substrates to trigger a cell cycle arrest. In cancer cells, this regulation may be faulty and cell division may proceed even in the presence of damaged DNA. We show here that the RSK kinase, often elevated in cancers, can suppress DSB-induced ATM activation in both Xenopus egg extracts and human tumor cell lines. In analyzing each step in ATM activation, we have found that RSK disrupts the binding of the MRN complex to DSB DNA. RSK can directly phosphorylate the Mre11 protein at Ser 676 both in vitro and in intact cells and can thereby inhibit loading of Mre11 onto DSB DNA. Accordingly, mutation of Ser 676 to Ala can reverse inhibition of the DSB response by RSK. Collectively, these data point to Mre11 as an important locus of RSK-mediated checkpoint inhibition acting upstream of ATM activation.</p><p>The phosphorylation of Mre11 on Ser 676 is antagonized by phosphatases. Here, we screened for phosphatases that target this site and identified PP5 as a candidate. This finding is consistent with the fact that PP5 is required for the ATM-mediated DNA damage response, indicating that PP5 may promote DSB-induced, ATM-dependent DNA damage response by targeting Mre11 upstream of ATM.</p> / Dissertation

The E3 ubiquitin ligase \(CRL4^{Cdt2}\) targets proteins for destruction during DNA replication and following DNA damage (Havens and Walter, 2011). Its substrates contain "PIP degrons" that mediate substrate binding to the processivity factor PCNA at replication forks and damage sites. The resulting PCNA-PIP degron complex forms a docking site for \(CRL4^{Cdt2}\), which ubiquitylates the substrate on chromatin. Several \(CRL4^{Cdt2}\) substrates are known, including Cdt1, multiple CDK inhibitors, Drosophila E2f1, human Set8, S. pombe Spd1, and C. elegans \(Pol\eta\) (Havens and Walter, 2011). An emerging theme is that \(CRL4^{Cdt2}\) targets proteins whose presence in S phase is toxic. Here, I used Xenopus egg extract to characterize a new \(CRL4^{Cdt2}\) substrate, thymine DNA glycosylase (TDG). TDG is a base excision repair protein that targets G-U and G-T mispairs, which arise from cytosine and 5-methylcytosine deamination (Cortazar et al., 2007). Thus, TDG may function in epigenetic gene regulation via DNA demethylation, in addition to its canonical DNA repair function. A yet unknown E3 ubiquitin ligase triggers TDG destruction during S phase (Hardeland et al., 2007). Understanding TDG proteolysis in S phase is relevant to the regulation of DNA replication, DNA repair, and epigenetic control of gene expression. I discovered that TDG contains a variant of the "PIP degron" consensus and that TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and degron-specific manner during DNA repair and DNA replication in Xenopus egg extract. I further characterized what features of TDG contribute to its proteolysis. Interestingly, I could not identify any defects during DNA replication or during Xenopus embryonic development in response to a non-degradable form of TDG. Additionally, I examined how interactions between \(CRL4^{Cdt2}\) and multiple subunits of the PCNA homotrimer contribute to \(CRL4^{Cdt2}\) function. In a popular model, PCNA functions as a "tool belt" on DNA, binding three separate proteins through its individual subunits to facilitate rapid exchange of DNA replication and repair proteins as they are needed on DNA. To address this model, I generated a single chain polypeptide with three PCNA subunits connected through flexible linker sequences. I used this tool to determine how multiple PCNA subunits contribute to \(CRL4^{Cdt2}\) function. I found that a single wildtype subunit is sufficient for modest destruction of the \(CRL4^{Cdt2}\) substrate Cdt1, but complete Cdt1 destruction requires two separate wildtype subunits. Additionally, a single subunit was sufficient for leading strand elongation, challenging the "tool belt" model during DNA replication. I also discuss implications and future use of the single-chain PCNA.

Aurora A (AurA) is an important mitotic kinase mainly studied for its involvement in cell cycle progression, centrosome maturation, mitotic spindle pole organization and bipolar spindle formation. It localizes to duplicated centrosomes and spindle microtubules (MTs) during mitosis where it regulates various factors participating in metaphase spindle formation. AurA is degraded late in mitosis suggesting that it might also have a function in anaphase. In this study we focused in understanding AurA function during anaphase in two different experimental systems. First, we kept AurA active in cycled Xenopus egg extracts and found that MTs maintained their mitotic organization longer throughout mitotic exit. We also observed chromosome segregation defects and problematic nuclear envelope formation. These observations indicate that AurA activity needs to be down-regulated for the transition from metaphase back to interphase. To get insights into the role of AurA during metaphase-anaphase transition we initially asked whether its kinase activity is still necessary for the maintenance of the metaphase spindle. We saw that the inhibition of AurA kinase activity in metaphase resulted to a collapse of the established metaphase spindle in HeLa cells. Indicating that AurA activity is necessary for the metaphase spindle maintenance. Then, we looked whether AurA kinase activity is still necessary during anaphase. We inhibited AurA at the onset of anaphase in Hela cells and found that anaphase spindles were smaller. We also observed that the MT structure responsible for anaphase spindle elongation, the central spindle, was defectively assembled and organized. Moreover, in cells where AurA was inhibited segregation of chromosomes was defective. These results indicate that AurA kinase activity is necessary for anaphase spindle elongation, central spindle assembly and organization and chromosome segregation. To understand further how AurA regulates anaphase spindle formation we looked known AurA substrates. We depleted TACC3, a known AurA substrate involved in MT formation earlier in mitosis and observed that TACC3 depletion phenocopied AurA inhibition. This indicates that TACC3 has a function in MT organization and chromosome segregation during anaphase and this function could possibly be regulated by AurA. In this study we have demonstrated that AurA activity is essential for metaphase spindle maintenance. We also found that during anaphase when AurA is either maintained active or inhibited MT organization is greatly affected and chromosome segregation is defective. Suggesting that AurA activity needs to be tightly controlled during anaphase for a correct completion of mitosis. / Aurora A (AurA) es una quinasa mitótica importante que se ha estudiado principalmente en su papel durante la progresión del ciclo celular, la maduración del centrosoma, la organización y la formación del polo y del huso mitótico. Durante la mitosis, AurA se localiza en los centrosomas duplicados y en los microtúbulos (MTs) del huso y se ha observado que regula varios factores que participan en la formación del huso mitótico. AurA se degrada al final de la mitosis indicando que pueda tener una función durante la anafase. En este estudio nos hemos centrado en la comprensión de la función de AurA durante la anafase en dos sistemas experimentales diferentes. En primer lugar, utilizando extractos de huevos de Xenopus hemos mantenido AurA activa durante la transición de metafase a anafase y hemos visto que los MTs del huso mitótico mantienen su organización durante más tiempo. También hemos observado que cuando AurA se mantiene activa existen defectos en la segregación cromosómica y la formación de la membrana nuclear. Esto indica que la actividad de AurA tiene un papel regulador sobre los MTs y la chromatina durante la transición de la metafase a la interfase. Para entender cual es la función de AurA durante la transición de metafase a anafase primero hemos estudiado si la actividad de la quinasa es necesaria para el mantenimiento del huso mitótico. Hemos visto que la inhibición de la actividad quinasa AurA resultó en el colapso del huso durante la metafase en células HeLa. Esto indica que la actividad de AurA es necesaria para el mantenimiento del huso mitótico de metafase. A continuación hemos analizamos si la actividad quinasa de AurA sigue siendo necesaria para la anafase. Para ello hemos inhibido AurA en células Hela al inicio de la anafase. En estas condiciones los husos de la anafase son más pequeños y la estructura de los MTs responsable del alargamiento del huso mitótico durante la anafase, el huso central, se organiza defectuosamente. Además, se encontraron errores durante la segregación de los cromosomas. Estos resultados indican que la actividad quinasa de AurA es necesaria para el alargamiento del huso durante la anafase y la organización y segregación cromosómica. Para entender el mecanismo de la función de AurA durante la anafase hemos estudiado a sustratos de AurA. Al estudiar TACC3 , un sustrato conocido de AurA que participa en la formación de MTs en las fase iniciales de la mitosis hemos encontrado que su eliminación de células HeLa produce el mismo fenotipo que la inhibición de AurA. Esto indica que TACC3 tiene una función en la organización de MT y la segregación de cromosomas durante la anafase y que esta función podría estar regulada por la quinasa AurA. En este estudio hemos demostrado que la actividad quinasa de AurA es esencial para el mantenimiento del huso mitótico. También hemos encontrado que durante la anafase cuando la quinasa AurA se mantiene activa o se inhibe la organización de los MTs del huso mitótico se ve muy afectada y los cromosomas se segregan defectuosamente. Por tanto los resultados de este estudio indican que la actividad quinasa de AurA está estrechamente controlada durante la anafase para el correcto cumplimiento de la mitosis.