Two conserved protein kinases, Chk1 and Chk2, are activated in response to genotoxic stress and mediate multiple cell cycle checkpoint mechanisms that ensure genomic integrity. The establishment of mitotic checkpoint delay in response to DNA damage or incompletely replicated DNA is conventionally thought to be accomplished through inhibition of the cyclin-dependent kinase, Cdc2. Both Chk1 and Chk2 have the potential to operate in this pathway. Therefore, the initial aim of this study was to investigate the relative requirement of Chk1 and Chk2 for mitotic delay mechanisms triggered by DNA damage and DNA replication arrest in avian and human cells. These studies demonstrated that Chk1 is the principal regulator of the G2/M checkpoint with a direct role in the establishment and maintenance of the mitotic delay in response to DNA damage. Chk1 was also found to be required for the S/M checkpoint in response to DNA replication arrest; however, detailed analysis indicated that its role is to maintain rather than initiate this checkpoint, as cells lacking functional Chk1 can initially delay mitosis for many hours before they enter a premature mitosis with unreplicated DNA. In avian cells, mitotic phosphorylation of cyclinB2 was found to be mediated by cyclin dependent kinases and suppressed by checkpoint signalling. However, accumulation of potentially active phospho-cyclinB2/Cdc2 complexes was observed during the initial mitotic delay in the absence of functional Chk1, suggesting that other factors apart from the conventionally known mechanisms can restrain mitotic Cdc2 activity. In addition, avian cells were able to delay mitosis effectively during replication arrest in the presence of the ATM/ATR inhibitor caffeine, further emphasizing the possibility of mitotic delay mechanisms that operate independently of ATM/ATR and Chk1. Furthermore, this study revealed that endogenous Cdc6 accumulates in a Chk1-dependent manner during replication arrest. To test whether Cdc6 might function upstream or downstream of Chk1 in the replication checkpoint pathway, Cdc6 was ectopically expressed in both checkpoint-proficient and checkpoint-deficient Chk1-depleted cells. The results from these intervention experiments give preliminary evidence that places Cdc6 downstream of Chk1 in the S/M checkpoint response. The ability of cells to delay the onset of mitosis while DNA replication is stalled independently of ATM/ATR/Chk1 is consistent with the general idea of an inherent relationship between the process of DNA replication and mitosis. The replication machinery might be able to signal either normal DNA replication in progress or the presence of stalled replication structures and thereby intrinsically link the successful completion of DNA synthesis to progression into mitosis.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:495181 |
| Date | January 2008 |
| Creators | Oehler, Verena |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/148/ |
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