| Περίληψη: | In order to maintain genomic integrity the cells have mechanisms ensuring that DNA is replicated faithfully and only once in each cell cycle. Genomic instability can result from aberrant replication and leads to tumorigenesis. Therefore, DNA replication is under strict regulation. Licensing DNA for replication is a regulatory mechanism ensuring faithful and only once per cell cycle duplication of the genome. The pre-Replicative Complex - pre-RC- is formed after passage through mitosis, onto ORC-bound origins of replication with the sequential recruitment of the licensing factors Cdt1, Cdc6 and the Mini Chromosome Maintenance 2-7 - MCM2-7 helicase complex. Once the MCM2-7 complex is loaded, DNA is licensed for replication in the subsequent S phase. In order to avoid re-replication, the protein levels of the licensing factors Cdt1 and Cdc6 are strictly regulated throughout the cell cycle.
In our previous work we were able to monitor the behavior of MCM proteins in live human cells, with the use of a real-time in vivo licensing assay and demonstrated that only a small proportion of MCM complexes are loaded upon mitotic exit, while the majority of MCM loading onto chromatin occurs in late G1, just prior to S phase onset (Symeonidou IE et al, 2013).
To further characterize the loading of MCM proteins onto chromatin during G1 phase, we analyzed the association of GFP-MCM4 with chromatin, using Fluorescence Recovery After Photobleaching (FRAP) analysis, in MCF7 breast cancer cells. We observed that loading of GFP-MCM4 onto chromatin occurs in two distinct waves, upon mitotic exit, in early G1 where a small fraction interacts with chromatin and in late G1, prior to S phase entry where full loading takes place. Analysis of the licensing factors showed that Cdc6 expression closely mirrors the waves of loading of MCM complexes. Subsequently, we used Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss in Photobleaching (FLIP) to assess the timing of MCM loading onto chromatin after depleting or ectopically expressing Cdc6 at specific cell cycle phases. Depletion of Cdc6 in late G1 showed that Cdc6 re-accumulation is essential for the wave of MCM loading at the G1/S phase transition. On the other hand, ectopic expression of Cdc6 in early G1, when it is normally absent, is sufficient to drive full MCM loading earlier in the cycle. These data suggest that Cdc6 levels direct the timing and extent of MCM loading during G1.
Since accurate DNA replication licensing is critical for the cells, we were interested in examining whether ablation of this additional loading step in late G1 would affect DNA replication in the subsequent S phase as well as overall progression into the cell cycle. We showed that under these conditions the cells undergo normal replication and progression into the cell cycle, however, when the cells were subjected to replication stress, they accumulated in S phase with increased DNA damage. DNA fiber analysis also revealed that under replication stress conditions the cells were unable to fire the full complement of dormant origins. These data suggest that the additional MCM loading wave in late G1 protects the cells from replication stress, by licensing dormant origins. We were also interested in investigating whether full MCM loading in late G1 would be linked with timing of early origins firing, however we weren’t able to identify such a correlation, although this hypothesis needs further investigation.
To better understand the properties of an overall rapid cell cycle with a very short G1 phase, we analyzed the cell cycle and the DNA replication licensing process in mouse embryonic stem cells, which posses such characteristics. We observed that Cdt1 is expressed upon mitotic exit and in G1 phase of mESCs and MCM2 associates with chromatin from G1 phase and upon entry into S phase exhibits the characteristic early-middle-late S phase expression patterns, suggesting that the licensing process in mESCs resembles somatic cells.
Maintenance of genome stability is of great importance in mESCs, since they can potentially give rise to a whole organism. Genetic lesions caused by exogenous or endogenous DNA damage can be proven disruptive for tissue and organismal homeostasis. We therefore also examined mESCs response following UV-C irradiation, at different phases of their cell cycle. We showed that mESCs are capable of arresting their cell cycle at G1/S, even though they are reported to lack restriction point. Additionally, they are able to rapidly proteolyse Cdt1, post damage in G1 phase, a feature that somatic cells also possess, while MCM2 chromatin loading exhibited an irregular phenotype, possibly due to arrest of the cell cycle. Irradiation of mESCs in mitosis revealed stabilization in Cdt1 expression, which occurred through its phosphorylation by MAP kinases, a pathway observed also in somatic cells, suggesting that similar regulatory pathways exist in pluripotent cells to maintain genomic integrity.
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