<p>To ensure genomic integrity, the genome must be accurately duplicated once and only once per cell division. DNA replication is tightly regulated by replication licensing mechanisms which ensure that origins only initiate replication once per cell cycle. Disruption of replication licensing mechanisms may lead to re-replication and genomic instability. </p><p>DNA licensing involves two steps including the assembly of the pre-replicative compelx at origins in G1 and the activation of pre-RC in S-phase. Cdt1, also known as Double-parked (Dup) in <italic> Drosophila Menalogaster </italic>, is a key regulator of the assembly of pre-RC and its activity is strictly limited to G1 by multiple mechanisms including Cul4<super>Ddb1</super> mediated proteolysis and inhibitory binding by geminin. Previous studies have indicated that when the balance between Cdt1 and geminin is disrupted, re-replication occurs but the genome is only partially re-replicated. The exact sequences that are re-replicated and the mechanisms contributing to partial re-replication are unknown. To address these two questions, I assayed the genomic consequences of deregulating the replication licensing mechanisms by either RNAi depletion of geminin or Dup over-expression in cultured Drosophila Kc167 cells. In agreement with previously reported re-replication studies, I found that not all sequences were sensitive to geminin depletion or Dup over-expression. Microarray analysis and quantitative PCR revealed that heterochromatic sequences were preferentially re-replicated when Dup was deregulated either by geminin depletion or Dup over-expression. The preferential re-activation of heterochromatic replication origins was unexpected because these origins are typically the last sequences to be duplicated during normal S-phase. </p><p>In the case of geminin depletion, immunofluorescence studies indicated that the re-replication of heterochromatin was regulated not at the level of pre-RC activation, but rather due to the restricted formation of the pre-RC to the heterochromatin. Unlike the global assembly of the pre-RC that occurs throughout the genome in G1, in the absence of geminin, limited pre-RC assembly was restricted to the heterochromatin. Elevated cyclin A-CDK activity during S-phase could be one mechanism that prevents pre-RC reassembly at euchromatin when geminin is absent. These results suggest that there are chromatin and cell cycle specific controls that regulate the re-assembly of the pre-RC outside of G1.</p><p>In contrast to the specific re-replication of heterochromatin when geminin is absent, re-replication induced by Dup over-expression is not restricted to heterochromatin but rather includes re-activation of origins throughout the genome, although there is a slight preference for heterochromatin when re-replication is initiated. Surprisingly, Dup over-expression in G2 arrested cells result in a complete endoreduplication. In contrast to the ordered replication of euchromatin and heterochromatin during early and late S-phase respectively, endoreduplication induced by Dup over-expression does not exhibit any temporal order of replication initiation from these two types of chromatin, suggesting replication timing program may be uncoupled from local chromatin environment. Taken together, these findings suggest that the maintenance of proper levels of Dup protein is critical for genome integrity.</p> / Dissertation

Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren. / Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.

PCNA est une protéine essentielle qui intervient dans de nombreux mécanismes cellulaires et qui possède de nombreuses modifications post-traductionnelles (MPTs) dont les fonctions de certaines, restent encore inconnues. Afin d’étudier la fonction de ces MPTs, nous avons développé un nouvel outil génétique permettant in cellulo, de substituer la protéine endogène PCNA par une version mutée de la protéine appelée version de complémentation. La technique consiste à cotransfecter des cellules en culture avec deux types de plasmides. Un premier plasmide permet l’invalidation du gène de PCNA endogène dans les cellules transfectées par le système CRISPR-Cas9. Le deuxième plasmide dit de complémentation permet l’expression d’une forme mutée de PCNA. Sur l’ensemble d’une banque de mutants testés, deux mutants de PCNA se sont avérés être létaux (D122A et E124A). Nous avons démontré que ces deux sites sont impliqués dans l’initiation d’une voie de dégradation ubiquitine dépendante CRL4Cdt2 essentielle pour la mise en place de la protéolyse d’un cocktail de protéines (cdt1, p21, set8) durant la phase S. Nous avons démontré que les cellules mutantes pour PCNA (D122A et E124A) accumulent la protéine p21. Ce défaut de dégradation de p21 provoque alors des évènements de re-réplication menant à terme à la mort des cellules mutantes. / PCNA is an essential protein that is involved in many cellular mechanisms and has many post-translational modifications (PTMs). The functions of some PTMs, still remain unknown. In order to study the function of these PTMs, we have developed a new genetic tool allowing, in cellulo, the substitution of endogenous PCNA protein with a mutated version of the protein named complementation version. The technique involves cotransfection of the cells in culture with two types of plasmids. A first plasmid allows invalidation of the endogenous PCNA gene in transfected cells by the CRISPR-Cas9 system. The second plasmid, named complementation plasmid allows the expression of a mutated form of PCNA. In the whole bank of tested mutants, two PCNA mutants were found to be lethal (D122A and E124A). We have demonstrated that these two sites are involved in the initiation of an ubiquitin-dependent protein degradation CRL4Cdt2 pathway essential for the proteolysis of a protein cocktail (cdt1, p21, set8) during the S phase. We demonstrated that PCNA mutant cells (D122A and E124A) accumulate p21 protein. This lack of degradation of p21 then causes re-replication events leading ultimately to the mutant cells death.