| Περίληψη: | Eukaryotic sizable genomes start their replication by simultaneous activation of multiple, dispersed origins, which fire stochastically during the S phase. Initiation of DNA replication from each origin is a two-step process, which involves loading of the MCM replicative helicases in an inactive state (licensing) and activation of the helicase at a later step (firing). Licensing of DNA replication is pivotal for ensuring that each origin will fire only once during the S phase of the cell cycle, providing a molecular memory for which origins have been activated, and thus should not be re-activated until the next cell cycle, and which not. CDC6/Cdc18 and Cdt1 are the two evolutionarily conserved from yeast to mammals licensing factors that load the MCM2-7 complexes onto the DNA forming the pre-Replicative complexes, that will be activated towards the G1/S transition due to CDK activity increase. The significance of the licensing process for maintenance of genome stability is evident, since its deregulation is accompanied by profound DNA damage, genetic instability, and loss of viability, thus being considered as a source of genotoxic stress. In fission yeast, Cdc18 overexpression is sufficient to overcome the ‘only once per cell cycle’ origin licensing rule and induces re-replication, the generation of replicative bubbles within bubbles, which is known to cause fork collision and DNA breakage. In humans, CDC6/CDT1 overexpression has been reported in pre-malignant cells, highlighting their role as oncogenes. This thesis was dedicated to investigating cellular/molecular mechanisms that may have evolved to early sense aberrations in the licensing system, promoting early responses to safeguard genome stability. To this end, we focused on the ribosomal DNA, an evolutionarily conserved genomic locus with multiple, densely spaced origins of replication. We speculated that this locus, due to several innate characteristics with most prominent the accumulation of many origins in a defined space, would be extra-sensitive in re-replication events. We set out to assess this hypothesis by utilizing the model organism fission yeast as an experimental system. Our data show that the rDNA copy number gets destabilized from an early timepoint upon induction of Cdc18 overexpression, before an established amplification hotspot gets increased. Moreover, the nucleolus organized around the rDNA accumulates phospho-H2A and Rad22 foci, two-established DSB markers even when mild re-replication is induced. In the second part of this thesis, we analyzed checkpoint controls elicited by Cdc18 overexpression. By combining experiments with wt Cdc18/N-terminally truncated Cdc18 in a rad3+/rad3Δ background, we show that Rad3-independent and Rad3-dependent pathways, initiated by Cdc18 O/E, act in synergy to achieve robust mitotic block even under mild re-replication, hence outlining Cdc18 as a master protector of genome integrity.
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