In eukaryotes, DNA is replicated once and only once per cell cycle from hundreds of replication initiation sites (origins) across the genome. This process is strictly controlled as mis-regulation can cause replication stress and genome instability, characteristics of cancer. S phase Cyclin-dependent kinase (S-CDK) plays a dual role in this regulation both by restricting helicase loading (licensing) to G1 phase, and by activating origin firing in S phase. The essential S-CDK phosphorylation of the limiting initiation proteins, Sld2 and Sld3, is sufficient for CDK’s role in replisome activation in Saccharomyces cerevisiae. This modification permits a cascade of protein recruitment to origins that completes replisome assembly, however, Sld2 and Sld3 do not travel with the replisome during replication elongation. It is not known whether the release of these factors from origins is phospho-dependent or whether this dissociation is important for replication control. In this thesis I investigate the observation that Sld2 and Sld3 are specifically, actively dephosphorylated in S phase. I identify PP2A-Rts1 and PP2A-Cdc55, alongside PP4, as the phosphatases responsible for Sld3 dephosphorylation and partially of Sld2. To overcome the pleiotropy of these phosphatases hindering S phase analysis, I identify the Sld3-PP2A-RTS1 interaction site and mutation of this site affects Sld3 dephosphorylation in S phase and at mitotic exit. Conditionally disrupting the S phase dephosphorylation of Sld3 results in impaired genome duplication and is lethal. Replication profiling and helicase chromatin immunoprecipitation (ChIP) show that this is due to a global defect in replication initiation. Adding recombinant PP2A-Rts1 to a reconstituted yeast replication system is sufficient to enhance DNA replication. Finally, I use ChIP-seq and genetics to test models for the mechanisms by which PP2A acts as a novel positive regulator of DNA replication initiation. This work has revealed that the dynamic turnover of Sld2 and Sld3 phosphorylation in S phase is essential. It appears that, like CDK, PP2A plays a critical dual role in preventing inappropriate replication initiation outside of S phase and stimulating it in S phase. With the conservation of this phenomenon to metazoa showing promise, this work should incentivise further studies to uncover the potential of PP2A phosphatases in differential S phase length control and as targets for cancer therapy.