BACKGROUND: Telomeres are the DNA sequences at the end of chromosomes that are made up of highly repetitive sequences to protect the ends of chromosomes from damage. Telomeres shorten with each round of DNA replication eventually causing cellular senescence. Many cancer cells are able to overcome or evade senescence by elongating their telomeres. Alternative lengthening of telomeres (ALT) is a recombination based mechanism used by cancer cells to maintain telomere length. Evidence supports that unresolved replication stress at telomeric DNA promotes telomere elongation. Given the role of RAD54 in recombination genome wide, we were interested in investigating whether RAD54 is contributing to ALT telomere maintenance. OBJECTIVE: To define the role of RAD54 in ALT telomere maintenance. METHODS: Several different known ALT cell lines and non-ALT cell lines were examined using wet-lab techniques. Combined immunofluorescence and DNA FISH (IF-FISH) was used to visualize co-localization between RAD54 and telomeres, siRNA was used to deplete specific mRNA, and western blots were used to confirm these knockdowns. RESULTS: IF-FISH showed enrichment of RAD54 at the telomeres in ALT cells as compared to non-ALT cells. There was a decrease in incorporation of the synthetic nucleotide EdU in the absence of RAD54, indicating a decrease in DNA synthesis. No change was seen in the recruitment of RAD51, a recombinase, to telomeres in the absence of RAD54. There was a significant increase in MUS81 colocalization to ALT telomeres in the absence of RAD54 and an increase in the number of ultra-fine anaphase bridges. CONCLUSIONS: Using combined immunofluorescence and DNA FISH, we found enrichment of RAD54 at the telomeres in ALT cells as compared to non-ALT cell lines. Furthermore, RAD54 is predominantly found at ALT telomeres in ALT-associated promyelocytic leukemia (PML) bodies (APBs), which are nuclear condensates containing telomeres and many repair proteins. APBs are thought to be sites of active recombination, mediated by the recombinase RAD51. We can monitor recombination using EdU incorporation events at telomeres. RAD54 promotes DNA synthesis events at ALT telomeres, as measured through EdU incorporation. No change was found in RAD51 recruitment to telomeres in the absence of RAD54, indicating that RAD54 is not required for RAD51 mediated synapsis. By over-expressing RAD54 mutants, we found that sites of DNA synthesis, that are thought to be elongation events at ALT telomeres, are dependent on the ATPase and branch migration activities of RAD54. These results suggest that RAD54 is promoting telomere elongation by mediating the migration of the branched DNA structures formed at telomeres during recombination. When RAD54 is depleted, we found an increase in recruitment of the nuclease MUS81 to ALT telomeres, suggesting that in the absence of branch migration, ALT telomeres are cleaved to resolve recombination intermediates. Together, these data demonstrate a crucial role for RAD54 in promoting ALT mediated telomere elongation through resolving homologous recombination intermediates via branch migration.

Telomeres are repetitive DNA sequences found at the ends of eukaryotic chromosomes that help maintain genome stability. Telomeres shorten every time a cell divides, eventually inducing replicative senescence. To gain replicative immortality cancer cells establish mechanisms to maintain telomere length over many cell divisions. Around 10% of cancers do this using a recombination-based pathway called the Alternative Lengthening of Telomeres (ALT). ALT resembles a specific type of homology-directed repair called break-induced replication (BIR). Through this body of work, we aimed to better understand both the genetics underlying ALT positive cancers and the mechanistic basis of ALT. ALT positive cancers frequently carry loss of function mutations in the genes for ATRX/DAXX, which function to regulate heterochromatin. Recently, we identified a novel chromosomal fusion event in ALT positive osteosarcoma causing defects in DAXX function. Additionally, we identified several osteosarcoma tumors with wild-type ATRX/DAXX that had abnormalities in SLX4IP or SMARCAL1, proteins recently shown to regulate the ALT pathway. These data suggest that a more thorough understanding of the ALT mechanism may reveal additional factors that are defective in ALT positive tumors. Building on this, we aimed to further define the mechanism of ALT by investigating the DNA translocase RAD54 in the ALT pathway. During BIR, a broken DNA strand invades a homologous template, forming a structure called a displacement loop (D-loop) where a strand of template DNA is displaced to allow base pairing between the broken DNA strand and the homologous template. The D-loop recruits DNA polymerases, leading to extension and repair of the broken DNA strand. RAD54 is known to regulate both the formation and resolution of D-loops. In this work, we found that RAD54 promotes elongation at ALT telomeres by mediating branch migration and dissolution of the D-loop. D-loops formed at ALT telomeres must be resolved before mitosis to prevent the formation of ultra-fine anaphase bridges. These data demonstrate that by mediating D-loop migration RAD54 plays an important role in both promoting telomere elongation and maintaining genome stability in ALT cells. Together this body of work represents advances in defining both the genetic and mechanistic basis of ALT. / 2021-01-30T00:00:00Z

La Recombinaison Homologue (RH) est une des voies majeures, hautement fidèle, de réparation des cassures double brin de l’ADN et du redémarrage des fourches de réplication arrêtées ou bloquées. La RH utilise une séquence homologue pour réparer avec précision l'ADN. Elle est essentielle pour le maintien de la stabilité des génomes dans tous les organismes et également pour assurer la transmission et l'échange de l'information génétique pendant la méiose. L'étude mécanistique de la RH est importante pour comprendre l'instabilité génétique, la perte d'hétérozygotie, les aberrations chromosomiques, la mort cellulaire et la cancérogenèse associée à une RH déficiente. Les étapes clés de la RH et les protéines impliquées sont très conservées dans toutes les espèces. Chez Saccharomyces cerevisiae, la recombinase Rad51 forme un filament présynaptique avec l’ADNsb qui est capable de rechercher les homologies de séquences dans tout le génome, en partenariat avec d'autres partenaires protéiques. Une fois l'homologie identifiée, une structure de D-loop (pour Displacement loop) est formée pour favoriser l'échange de brins. Le moteur moléculaire Rad54 assiste Rad51 dans la formation de la D-loop. Son rôle dans la recherche d'homologie et la formation des complexes synaptiques, avant mêle la formation de la D-loop reste un sujet de débat. Cette thèse porte sur mes travaux d’étude in vitro des mécanismes de formation de la D-Loop, en utilisant des protéines de la RH purifiées Rad51 et Rad54 avec d'autres partenaires protéiques et des substrats d'ADN synthétisés, mimant les structures de la RH. J'ai utilisé la microscopie électronique (ME) pour visualiser directement l'ADN et les complexes ADN-protéines intervenant au cours de la formation de D-loop in vitro avec Rad51, Rad54 et un mutant de Rad54. Ces approches d’imagerie, combinées à la biochimie suggèrent que Rad54 est crucial pour la recherche d'homologie et la formation du complexe synaptique, avant la formation de la D-loop, dans une coopération étroite avec Rad51. J'ai également montré que les paralogues de Rad51, Rad55-Rad57, stimulent la formation de la D-loop et que cet hétérodimère présente une activité ATPase dix fois plus forte que Rad51. Par ailleurs, j'ai également développé d'autres outils méthodologiques en ME et en microscopie à force atomique à haute vitesse (HS-AFM) pour mieux caractériser différents intermédiaires de la RH. / Homologous recombination (HR) is a major high-fidelity DNA repair pathway of double-stranded breaks and recovery of stalled and collapsed replication forks. HR uses a homologous template to accurately repair DNA that is essential for maintaining genomic stability in all organisms and to ensure the transmission and exchange of the genetic information during meiosis. The importance of HR study is highlighted by genetic instability, loss of heterozygosity, chromosomal aberrations, cell death and carcinogenesis associated with a defected HR. The key recombinational stages and proteins are well conserved throughout species. In Saccharomyces cerevisiae, the Rad51 recombinase forms a presynaptic filament with ssDNA that along with other protein partners is able to search for homology within the entire genome. Once homology is identified, a Displacement-loop (D-loop) is formed to promote strand-exchange. The Rad54 molecular motor assists Rad51 in the D-loop formation, and it is still a matter of debate whether it also plays a key role in homology search and synaptic complex formation, prior to D-loop. This dissertation covers my in vitro assays using purified key HR proteins Rad51 and Rad54, other protein partners and designed DNA substrates, mimicking HR structures.I used electron microscopy (EM) to directly visualize the HR DNA and DNA-protein complexes generated by D-loop in vitro assay with Rad51, Rad54 and a Rad54 mutant, and these studies combined with biochemistry suggest Rad54 is crucial to homology search and synaptic complex formation, prior to D-loop formation, in a tight intercooperation by Rad51 and Rad54. In a multiprotein system, I also showed the Rad51 paralogs Rad55-Rad57 stimulate the D-loop formation and that this heterodimer presents a ten times stronger ATPase activity than Rad51. I also developed other EM and high speed atomic force microscopy (HS-AFM) methodological tools to characterize other HR intermediates.