Abstract

Eukaryotic DNA replication relies on a tight two-step regulation to maintain genome stability and ensure that the genome is copied precisely once during each cell cycle. The first step – origin licensing – is restricted to late M and G1 phase of the cell cycle, whereas the second step – origin firing – is restricted to S, G2 and early M phase. This strict temporal separation of licensing and firing prevents uncontrolled over-replication, which is associated with hallmarks of genome instability such as gene amplifications or gross chromosomal rearrangements that are often observed during early stages of tumorigenesis. However, it is unclear how strict control is achieved on the molecular level at the transitions between licensing and firing. While the regulation of licensing factors has been studied in detail, much less is known about the regulation of firing factors. Particularly, it is unclear how firing factors are inactivated in M phase and how deregulation of firing factors causes genome instability. To address these gaps in our knowledge, we have established an experimental setup to monitor the dynamic regulation of replication proteins throughout the cell cycle in budding yeast cells. Our data demonstrate that at the transition in M phase firing is inactivated before licensing is re-activated, thereby generating an intermittent gap phase. Early inactivation of origin firing is mediated by precisely timed degradation of firing factor Sld2. We decipher the underlying degradation pathway involving four kinases and two ubiquitin ligases and demonstrate that preventing rapid degradation of Sld2 in M phase shortens the gap phase between firing and licensing. Importantly, such shortening of the gap in M phase results in significant levels of genome instability when combined with other replication mutants. Moreover, we investigate the cellular consequences of deregulated origin firing. In particular, we study deregulated origin firing in G1 phase and find that cellular DNA surveillance mechanisms such as the DNA damage checkpoint appear to be blind to this type of problem. Even after deregulated replication initiation in G1 phase, cells commit to another full round of DNA replication in S phase. Consequently, we observe strong induction of DNA damage in late S phase, which ultimately activates DNA damage signaling after bulk replication is finished. We identify factors required for this checkpoint response and characterize replisomes under over-replication conditions using mass spectrometry and next-generation sequencing. Taken together, our work reveals new regulatory controls of DNA replication and establishes gap phases as integral elements of replication control, which allow safe transitions between origin licensing and origin firing.