Maintenance of the spindle assembly checkpoint by PLK1 and CDC7 kinases and characterisation of cell lines carrying mutations in the genes coding for the CDC7 regulatory subunits
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The Spindle Assembly Checkpoint (SAC) ensures that accurate chromosome segregation occurs, by preventing progression into anaphase until all kinetochore-microtubule attachments are stable and bi-polar. During a mitotic arrest, induced by microtubule targeting drugs, the weakening of the SAC allows cells to progress through the cell cycle without chromosome segregation occurring. PLK1 plays a major role in mitosis and has recently emerged as a player in establishing and maintaining a robust SAC. However, mechanistically, the role of PLK1 in the SAC is not fully understood. CDC7 kinase is essential for the initiation of DNA replication in eukaryotic organisms. In humans, it requires the binding of a regulatory subunit, either DBF4 or DRF1, for its activity, which persists from S-phase until anaphase. To date, there is limited knowledge about the distinct functions of DBF4 and DRF1. Furthermore, the role for CDC7 kinase in mitosis in humans has not been established. In this thesis, the role of PLK1 in SAC maintenance was assessed. PLK1 was found to be required for maintenance of Aurora B and its activity at the kinetochores. PLK1 and Aurora B cooperate to maintain a robust SAC but PLK1 does not cooperate with MPS1 or Haspin during checkpoint maintenance. It was found that CDC7 is an active kinase during mitosis where it cooperates with PLK1 in SAC maintenance. Using genetically edited cell lines, this study has revealed that a deletion in DBF4 causes an accumulation of cells in S-phase, and defective phosphorylation of the replicative DNA helicase subunit, MCM2. These results indicate that PLK1 is directly involved in maintaining efficient SAC signalling and provide evidence for a novel role of CDC7 kinase in mitosis. Preliminary evidence suggests that DBF4 is the main CDC7 kinase regulatory subunit that is responsible for efficient S-phase progression and for phosphorylating MCM2.
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