Mechanisms of genomic stability maintenance in fission yeast

DNA replication consists of two distinct steps: the licensing step during the G1 phase and the initiation step at the G1 to S transition point. During licensing, two inactive complexes of the replicative helicases, MCMs, are loaded onto all the potential origins of replication (ORIs), through the combined action of Cdt1 and Cdc18. In a next step, a small subset of the licensed origins is activated in order to achieve timely duplication of the genome in the timeframe of S. The subset of origins activated in each cell cycle constitute the replication program of the cell. Despite the fact that origin selection is characterized by stochasticity, there are several parameters affecting the process and among them, nuclear architecture and the 3D genome structure are critical. In the present study, we set out to investigate the implication of the latter parameters in the replication program, using the model organism S. pombe. Combining genome-wide datasets regarding the exact position and the activation frequency per cell cycle (efficiency) for each origin with the 3D genome model in S. pombe, we noticed a spatial compartmentalization between efficient and inefficient origins, with the former presenting a position bias towards centromeres, which are clustered and anchored at a specific point of the nuclear membrane. Immunofluorescence experiments against IdU in wild type and mutant cells carrying unclustered centromeres (Csi1Δ), that allow capturing of early initiation events during replication, indicated that centromere mislocalization disrupts the spatial pattern of origin firing during early S. Next, in order to experimentally validate our initial, model-derived observation, efficient and inefficient ORIs, residing in all the three chromosomes, were tagged using the lacO-lacI system and their distance from centromeres in the 3D space of the nucleus was measured at the single cell level, employing confocal microscopy. The firing efficiencies of the respective origins were measured by Real-time qPCR and correlated to the distances. Our results experimentally confirmed a negative correlation between the ORI-centromere distance and the origin efficiency. In order to elucidate the molecular basis of this negative correlation, we investigated a possible, additional role for the master regulator of the replication program, Rif, in the 3D organization of the ORIs within the nucleus. To that end, the 3D distances of the tagged ORIs in respect to centromeres were measured for wild type and Rif1Δ cells. We noticed that, Rif1 selectively affects the positioning of Rif1-regulated ORIs, pointing to a differential role for the protein in chromatin organization. The temporal separation between licensing and initiation ensures the once and only once duplication of the genome and is achieved through the tight regulation over Cdt1 and Cdc18, which restricts their activity in the G1 phase. Deregulation of Cdt1 and Cdc18 leads to re-replication, a process generating multiple copies of specific parts of the genome. Another phenomenon related to increased copies of specific genomic segments are the Copy Number Gains (CNGs). In order to establish a cause and effect relationship between re-replication and CNGs, an inducible cdc18 overexpressing cell strain was exploited. Re-replication was induced and the incidence of CNG formation was monitored following specific genomic loci that are able to confer drug resistance when present in multiple copies. Our results indicated that, indeed, re-replication drives the formation of CNGs. Whole genome sequencing analysis of resistant clones indicated that CNGs are present in the form of Mb-long, extrachromosomal inverted repeats. Finally, to elucidate the DNA damage repair mechanisms that mediate the formation of CNGs upon re-replication induction, the incidence of re-replication induced CNGs was measured in mutant strains, bearing specific repair pathways inactivated. We noticed that the multiple copies generated by re-replication are converted into stable, inheritable genotypes through various Homologous Recombination-related repair pathways acting in a locus-specific manner. DNA replication is critical for cell survival. Therefore, elucidating both its regulation under normal conditions and its implications upon deregulation, could help us understand the molecular basis of related diseases and facilitate the development of more successful treatment methods.

Author dc:creator

• Ναθαναηλίδου, Πατρούλα

Contributors dc:contributor

• Λυγερού, Ζωή
• Nathanailidou, Patroula
• Ταραβήρας, Σταύρος
• Wu, Pei-Yun Jenny
• Σταθόπουλος, Κωνσταντίνος
• Ζαρκάδης, Ιωάννης
• Παπαχαντζοπούλου, Αδαμαντία
• Σπηλιοπούλου-Σδούγκου, Ίρις

• Schizosaccharomyces pombe
• DNA replication
• Replication licensing
• Rereplication
• Gene amplification
• Σχιζοσακχαρομύκητας
• Αντιγραφή
• Αδειοδότηση αντιγραφής
• Επαναντιγραφή
• Γονιαδιακή ενίσχυση

Statement dc:rights

• 12

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/10889/14479

OAI identifier oai:identifier

oai:nemertes.library.upatras.gr:10889/14479