Mutations in five protein components of the pre-replication complex (pre-RC) have been identified in patients diagnosed with Meier-Gorlin syndrome (MGS), a disorder of global extreme growth failure. ORC1, the most commonly mutated protein, is the largest of six subunits in the origin recognition complex which associates with genomic replication origins and initiates the assembly of the pre-RC during G1 of the cell cycle. Mutations in ORC1, therefore, are expected to perturb cell cycle progression resulting in lengthened cell cycle which leads to a reduction in overall cell number generated during embryogenesis. In this thesis I investigate the developmental and cellular consequences of mutations in ORC1. To address the role of ORC1 during mammalian development I utilised CRISPR/cas9 technology to generate an allelic series of Orc1 mutations in mouse embryonic stem cells (mESCs). Mouse embryos generated by tetraploid complementation assays, using hypomorphic Orc1-mESCs, indicates that impaired Orc1 function in mouse embryogenesis results in developmental delay and a reduction in embryo size. While, no change in proliferation dynamics was seen in Orc1-mESCs in vitro, despite the markedly shortened cell cycle in such cells. Similarly, no significant differences in cell cycle or proliferation rate were detectable in primary patient-derived fibroblasts and lymphoblastoid cell lines. In contrast, Orc1-deficient mouse embryonic fibroblasts had a lengthened cell cycle, as a result of a prolonged G1 phase, implicating the role of the replication licensing checkpoint in somatic cells in lengthening of the cell cycle. In addition, the impact of complete loss of ORC1 was investigated in the developing limb bud. Generation of an Orc1 conditional allele provided a tool to analyse how loss of ORC1 would alter cell cycle in the developing limb. ORC1 is required for embryonic development, and homozygous deletion is embryonic lethal. Mesoderm specific deletions in Orc1 in the developing limb bud using the Prx1-cre transgene resulted in complete loss of forelimb structures, and oligodactyl of the hind limb. Finally, a novel disease gene was identified in patients with MGS. Mutations were identified in 9 patients (7 families) with mutations in CDC45. CDC45 acts downstream of the pre-RC and is required for the formation of the pre-initiation complex, origin activation and fork elongation. As CDC45 acts in the same pathway as ORCs, CDC6 and CDT1, it was hypothesised that hypomorphic mutations in CDC45 might also result in MGS due to impaired DNA replication.

Em eucariotos, a origem de replicação é reconhecida por um complexo ORC, a proteína Cdc6 e outras proteínas. Nos tripanossomas, encontramos somente uma proteína similar a Orc1 e Cdc6, que chamaremos de Orc1/Cdc6. Nesta tese serão descritos os estudos realizados sobre Orc1/Cdc6 do Trypanosoma cruzi e do Trypanosoma brucei. As proteínas recombinantes dos tripanossomas apresentam atividade de ATPase e são capazes de substituir Cdc6 em ensaio de complementação em leveduras. A indução do silenciamento do gene de Orc1/Cdc6 por RNAi resulta em células anucleadas. Orc1/Cdc6 é expressa durante todo o ciclo celular das formas replicativas, permanecendo associada à cromatina. No caso do T.cruzi, Orc1/Cdc6 é expressa não só nas formas replicativas, mas também nas formas não replicativas. Nestas últimas, a proteína expressa não interage com o DNA, este resultado sugere que a ausência desta interação deve contribuir para ausência da duplicação do DNA nas formas infectivas do T. cruzi. / In eukaryotes, the replication origin is recognized by a complex ORC, Cdc6 and other proteins. The trypanosomes contain only one protein, we named it Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from T.cruzi (TcOrc1/Cdc6) and from T.brucei (TbOrc1/Cdc6) present ATPase activity, replaced yeast Cdc6 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T.brucei resulted in enucleated cells. Orc1/Cdc6 is expressed during the entire cell cycle and in all stages of the life cycle of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. This association is different among the stages from T. cruzi life cycle. In the non replicative ones, Orc1/Cdc6 does not interact with DNA. The lack of pre-replication machinery-DNA interaction in T. cruzi non-replicative stages might contribute to the absence of DNA replication in these stages.

N"alpha"-Acetylierung, eine der häufigsten eukaryontischen Proteinmodifikationen, wird von N-terminalen Acetyltransferasen (NATs) katalysiert. NatA, die bedeutendste NAT in Saccharomyces cerevisiae, besteht aus den Untereinheiten Nat1, Ard1 und Nat5, und ist am silencing, d.h. am Aufbau repressiver Chromatinstrukturen an Telomeren und den Paarungstyp-Loci HML und HMR beteiligt. Die vorliegende Arbeit demonstriert eine Rolle von NatA auch beim rDNA-silencing, und zeigt erstmals, dass die silencing-Faktoren Orc1 und Sir3 funktionell von der N"alpha"-Acetylierung durch NatA abhängen. Orc1, die größte Untereinheit des origin recognition complex (ORC), wurde in vivo durch NatA N"alpha"-acetyliert. Mutationen, die dies verhinderten, bewirkten eine starke telomerische Derepression. NatA wirkte genetisch über die ORC Bindungsstelle des HMR-E-silencers. Die artifizielle Bindung von Orc1 an HMR-E machte HMR-silencing NatA-unabhängig. Auch die synthetische Letalität von nat1"DELTA" orc2-1 Doppelmutanten wies auf eine funktionelle Verbindung zwischen NatA und ORC hin. Als weiteres NatA-Substrat wurde Sir3 identifiziert, dessen zelluläre Lokalisierung von NAT1 abhing. Die schwächeren silencing-Defekte der unacetylierten orc1 sir3 Doppelmutante im Vergleich zu nat1"DELTA" implizierten allerdings, dass noch weitere silencing-Proteine die N"alpha"-Acetylierung für ihre Funktion bedürfen. Weitere Ergebnisse dieser Arbeit belegen eine Funktion N-terminalen 100 Aminosäuren von Orc1 im silencing. Deletionen innerhalb dieses Bereichs erzeugten silencing-Defekte. Das Fehlen von 51 Aminosäuren vom N-Terminus von Orc1 unterbrach die Interaktion mit Sir1, verstärkte aber auch den silencing-Defekt von sir1"DELTA". Dies ergibt ein Model, in dem Orc1 neben Sir1 ein weiteres silencing-Protein rekrutiert, das zu seiner Bindung einen intakten, acetylierten N-Terminus von Orc1 benötigt. Zusammenfassend sprechen die Ergebnisse für eine Rolle der N"alpha"-Acetylierung durch NatA bei der Modellierung der Chromatinstruktur. / N"alpha"-acetylation, one of the most abundant eukaryotic protein modifications, is catalyzed by N-terminal acetyltransferases (NATs). NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1, Ard1 and Nat5 and is necessary for the assembly of repressive chromatin structures at the silent mating type loci and telomeres. This thesis shows that NatA also acts in rDNA repression and it provides the first direct evidence for the functional regulation of the silencing factors Orc1 and Sir3 by NatA-dependent N"alpha"-acetylation.Orc1, the large subunit of the origin recognition complex (ORC), was N"alpha"-acetylated in vivo by NatA. Mutations that abrogated this acetylation caused strong telomeric derepression. NatA functioned genetically through the ORC binding site of the HMR-E silencer. Direct tethering of Orc1 to HMR-E circumvented the requirement for NatA in silencing. The synthetic lethality of nat1"DELTA" orc2-1 double mutants further supported a functional link between NatA and ORC.Sir3 was also indentified as a NatA substrate. Its localization to perinuclear foci was NAT1 dependent. Unacetylated sir3 orc1 double mutants did not resemble the nat1"DELTA" silencing phenotype. Thus, we suggest that further silencing components require NatA-dependent N"alpha"-acetylation for their function. We further identified the N-terminal 100 amino acids of Orc1 to be important for silencing, since truncations within this region impaired silencing. The deletion of 51 amino acids from the Orc1 N-terminus interrupted the interaction with Sir1 and also reduced silencing in sir1"DELTA" strains. We thus propose that the silencing function of Orc1 is not restricted to Sir1 recruitment, but also comprises the interaction with another protein. The silencing function of this hypothesized interaction partner may depend on the N"alpha"-acetylation and integrity of the N-terminus of Orc1.In summary, we propose that N"alpha"-acetylation by NatA represents a protein modification that modulates chromatin structure in yeast.

Em eucariotos, a origem de replicação é reconhecida por um complexo ORC, a proteína Cdc6 e outras proteínas. Nos tripanossomas, encontramos somente uma proteína similar a Orc1 e Cdc6, que chamaremos de Orc1/Cdc6. Nesta tese serão descritos os estudos realizados sobre Orc1/Cdc6 do Trypanosoma cruzi e do Trypanosoma brucei. As proteínas recombinantes dos tripanossomas apresentam atividade de ATPase e são capazes de substituir Cdc6 em ensaio de complementação em leveduras. A indução do silenciamento do gene de Orc1/Cdc6 por RNAi resulta em células anucleadas. Orc1/Cdc6 é expressa durante todo o ciclo celular das formas replicativas, permanecendo associada à cromatina. No caso do T.cruzi, Orc1/Cdc6 é expressa não só nas formas replicativas, mas também nas formas não replicativas. Nestas últimas, a proteína expressa não interage com o DNA, este resultado sugere que a ausência desta interação deve contribuir para ausência da duplicação do DNA nas formas infectivas do T. cruzi. / In eukaryotes, the replication origin is recognized by a complex ORC, Cdc6 and other proteins. The trypanosomes contain only one protein, we named it Orc1/Cdc6. Here we show that the recombinant Orc1/Cdc6 from T.cruzi (TcOrc1/Cdc6) and from T.brucei (TbOrc1/Cdc6) present ATPase activity, replaced yeast Cdc6 in a phenotypic complementation assay. The induction of Orc1/Cdc6 silencing by RNA interference in T.brucei resulted in enucleated cells. Orc1/Cdc6 is expressed during the entire cell cycle and in all stages of the life cycle of trypanosomes, remaining associated with chromatin in all stages of the cell cycle. This association is different among the stages from T. cruzi life cycle. In the non replicative ones, Orc1/Cdc6 does not interact with DNA. The lack of pre-replication machinery-DNA interaction in T. cruzi non-replicative stages might contribute to the absence of DNA replication in these stages.

Les organismes cellulaires appartiennent à l'un des trois domaines du vivant : Archaea, Bacteria, Eucarya. Les Archaea sont des organismes unicellulaires avec un phénotype bactérien mais qui possèdent de nombreux caractères moléculaires eucaryotes. En particulier, la machinerie de réplication archéenne est une version homologue et simplifiée de celle des eucaryotes. Au cours de cette thèse, j'ai étudié la réplication de l'ADN chez les Archaea en combinant des approches in vitro et in silico.<br />Premièrement, j'ai essayé de purifier la protéine initiatrice de la réplication Cdc6/Orc1, sous une forme native, dans l'espoir de mettre au point le premier système de réplication de l'ADN in vitro chez les Archaea. Malheureusement, cette approche a été infructueuse en raison de l'instabilité et des propriétés d'agrégation de la protéine.<br />Deuxièmement, j'ai réalisé une analyse comparative du contexte génomique des gènes de réplication dans les génomes d'Archaea. Cette analyse nous a permis d'identifier une association très conservée entre des gènes de la réplication et des gènes liés au ribosome. Cette organisation suggère l'existence d'un mécanisme de couplage entre la réplication de l'ADN et la traduction. De manière remarquable, des données expérimentales obtenues chez des modèles bactériens et eucaryotes appuient cette idée. J'ai ensuite mis au point des outils expérimentaux qui permettront d'éprouver la pertinence biologique de certaines des prédictions effectuées.<br />Finalement, j'ai examiné la distribution taxonomique des gènes de la réplication dans les génomes d'Archaea afin de prédire la composition probable de la machinerie de réplication de l'ADN chez le dernier ancêtre commun des Archaea. Dans leur ensemble, les profils phylétiques des gènes de la réplication suggèrent que la machinerie ancestrale était plus complexe que celle des organismes archéens contemporains.