DNA Replication Crosstalk with the Cell Cycle
As the replisome acts to duplicate the genome, the preRC is disrupted and reassembly is prevented by various mechanisms with largely target Cdc6 and Cdt1. This ensures that replication initiation occurs only once at a given origin during a single cell cycle.
The p16-pRB-E2F axis regulates preRC assembly via transcriptional or post-transcriptional mechanisms in a cell cycle-dependent manner. During asynchronous growth, overexpression of p16ink4a blocks assembly (but not total protein levels) of the MCM complex onto chromatin, which inhibits downstream events in DNA replication such as recruitment of RPA and PCNA to chromatin. Once MCM is bound to chromatin in S-phase, the association is not disrupted by overexpression of p16ink4a. These effects of p16ink4a overexpression on MCM loading in cycling cells are pRB-dependent as they can not be recaitulated in the pRB-negative Saos-2 cell line unless transfected with Ad-pRBΔCDK (i.e. constitutively active).
During both asynchonous growth and during S-phase, pRB overexpression (but not p16ink4a overexpression) displaces PCNA from chromatin. Since pRB displaces chromatin-bound PCNA during S-phase, this supports evidence suggesting pRB controls DNA replication at the post-transcriptional level, beyond the influence of E2F-dependent transcriptional regulation.
The p16-pRB-E2F axis regulates preRC assembly via transcriptional or post-transcriptional mechanisms in a cell cycle-dependent manner. During asynchronous growth, overexpression of p16ink4a blocks assembly (but not total protein levels) of the MCM complex onto chromatin, which inhibits downstream events in DNA replication such as recruitment of RPA and PCNA to chromatin. Once MCM is bound to chromatin in S-phase, the association is not disrupted by overexpression of p16ink4a. These effects of p16ink4a overexpression on MCM loading in cycling cells are pRB-dependent as they can not be recaitulated in the pRB-negative Saos-2 cell line unless transfected with Ad-pRBΔCDK (i.e. constitutively active).
During both asynchonous growth and during S-phase, pRB overexpression (but not p16ink4a overexpression) displaces PCNA from chromatin. Since pRB displaces chromatin-bound PCNA during S-phase, this supports evidence suggesting pRB controls DNA replication at the post-transcriptional level, beyond the influence of E2F-dependent transcriptional regulation.