No description available.

DNA damage or replication stress during S-phase can activate the S-phase checkpoint which executes a variety of responses, such as the inhibition of origin firing and replication fork stabilisation. Deregulation of the S-phase checkpoint leads to genomic instability, which has been implicated in diseases such as cancer. In this thesis, I aimed to address whether the S-phase checkpoint is regulated outside of S-phase, and how the S-phase checkpoint targets its substrates in budding yeast. Although this checkpoint has thus far been associated exclusively with S-phase, it remains unknown whether its responses such as inhibition of origin firing can also occur in other phases of the cell cycle. To investigate this, the targets of the S-phase checkpoint for the inhibition of origin firing were analysed outside of S-phase upon DNA damage. Interestingly, I showed that the S-phase checkpoint effector kinase Rad53 phosphorylates its targets to inhibit origin firing outside of S-phase upon DNA damage when there is no replication. I then set out to test whether inhibition of origin firing by Rad53 outside of S-phase might be important for faithful DNA replication. Having shown that the checkpoint response is not specific for any cell cycle phases, I then tested how the specificity of Rad53 for its substrates might be determined. After demonstrating that the essential replication protein Cdc45 is required for Rad53 to phosphorylate the initiation factor Sld3, the key residues of Cdc45 necessary for Rad53 interaction were identified. A Cdc45 allele was produced by mutating the identified residues. This allele of Cdc45 is a separation-of-function mutant which prevents Sld3 phosphorylation upon DNA damage, but retains its function in DNA replication. Because Cdc45 travels with the replication fork, it is possible that Cdc45 also targets Rad53 to the replication fork to stabilise it upon replication stress. Overall, this thesis provides evidence that the S-phase checkpoint can function throughout the cell cycle and that Cdc45 targets Rad53 to some of its substrates, and possibly plays a role in replication fork stabilisation.

The CMG (Cdc45, MCM, GINS) helicase is required for cellular proliferation and functions to unwind double-stranded DNA to allow the replication machinery to duplicate the genome. Cancer cells mismanage helicase activation through a variety of mechanisms, leading to the potential for the development of novel anti-cancer treatments. Mammalian cells load an excess of MCM complexes that act as reserves for new replication origins to be created when replication forks stall due to stress conditions, such as drug treatment. Targeting the helicase through inhibition of the MCM complex has sensitized cancer cells to drugs that inhibit DNA replication, such as aphidicolin and hydroxyurea. However, these drugs are not used in the clinical management of cancer. We hypothesized that the effectiveness of the clinically relevant drugs gemcitabine and 5-FU against pancreatic cancer cells, and oxaliplatin and etoposide against colorectal cells, could be increased through co-suppression of the MCM complex. The oncogene c-Myc also leads to the mismanagement of CMG helicases in part due to a non-transcriptional role in overactivating replication origins and causing DNA damage. We sought to elucidate the mechanism by which Myc causes overactivation of CMG helicases. Herein we demonstrate that co-suppression of reserve MCM complexes in pancreatic or colorectal cancer cell lines treated with clinically applicable chemotherapeutic compounds causes significant loss of proliferative capacity compared with cells containing the full complement of reserve MCMs. This is in part due to an inability to recover DNA replication following drug exposure, leading to an increase in apoptosis. Targeting of Myc to genomic sites induced large-scale decondensation of higher order chromatin that was required for CMG helicase assembly and activation at reserve MCM complexes. The physiological mediators of Myc, GCN5 and Tip60, are required for the chromatin unfolding and Cdc45 recruitment. We conclude that depletion of the reserve MCM complexes causes chemosensitization of multiple human tumor cell types to several chemotherapeutic drugs used in the clinical management of human cancer. This argues for the development and use of anti-MCM drugs in combination with chemotherapeutic compounds, which has the potential to increase the therapeutic index of existing clinical compounds. We have also identified a previously unknown role for Myc in normal cell cycle progression whereby DNA replication initiation is regulated through the assembly and activation of CMG helicases on Myc-mediated open chromatin regions. Our results also provide new mechanistic insight into Myc oncogenic transformation in which overstimulation of DNA replication could result in genomic instability and provide an explanation for Myc driven oncogenic transformation.

No description available.

No description available.

No description available.