In all eukaryotes, it is essential that cells maintain a strict control over S phase to ensure that DNA replication occurs once, and only once, per cell division cycle. To achieve this, cells must promote DNA replication during the G1 phase and then repress DNA replication during the G2 phase. In the fission yeast Schizosaccharomyces pombe, two proteins required for the initiation of DNA replication, Cdcl8 and Cdt1, may be important for this control over DNA replication. Cdcl8 and Cdt1 are both upregulated in G1 and have an essential function in initiation, and are then efficiently downregulated in G2. In this thesis, I have tested whether the downregulation of these initiation factors is important to repress DNA synthesis in G2. I have found that the accumulation of high levels of Cdcl8 are sufficient to induce origin firing in G2 cells and drive re-replication in the absence of mitosis. This re-replication is potentiated by the co-expression of Cdt1, which may be mediated by the stabilisation of Cdc18 on chromatin by Cdt1. Biochemical characterisation of these re-initiation events revealed that certain aspects of this re-replication are similar to those observed in wild type S phase cells, such as the requirement for the MCMs and the cyclin-dependent kinase Cdc2. However, these cells fail to undergo complete and efficient replication of the chromosomes suggesting that Cdc18 and Cdt1 can override the normal controls that block initiation, but this mechanism does not fully mimic initiation in a G1 cell. The findings presented in this thesis have identified and characterised multiple, overlapping mechanisms that downregulate Cdc18 and Cdt1 in G2. If these mechanisms are disrupted and Cdc18 and Cdt1 are allowed to accumulate in G2, replication competence can be re-established and cells re-replicate. Therefore, Cdc18 and Cdt1 form the core of the control which licenses chromosomes for replication
