Origin Association of Sld3, Sld7, and Cdc45 Proteins is a Key Step for Determination of Origin- Firing Timing

Chromosomal DNA replication in eukaryotes initiates from multiple origins of replication and because of this multiplicity, activation of replication origins is likely to be highly coordinated; origins fire at characteristic times, with some origins firing on average earlier (early-firing origins) and others later (late-firing origins) in S phase of budding yeast. However, the molecular basis for such temporal regulation is poorly understood. Here, we show that origin association of low abundant replication proteins, Sld3, Sld7, and Cdc45, is the key to determining the temporal order of origin firing. These proteins form a complex and associate with the 'early'-firing origins in G1 phase in a manner that depends on Dbf4-dependent kinase (DDK), which is essential for the initiation of DNA replication. An increased dosage of Sld3, Sld7 and Cdc45 allows the late-firing origins to fire earlier in S phase. Additionally, an increased dosage of DDK also allows the late-firing origins to fire earlier. Thus, the limited association between origins and Sld3-Sld7-Cdc45 is a key step for determining the timing of origin firing. Our results unveil not only the molecular basis of temporal regulation of origins, but also the novel level of the control of origins in the initiation of DNA replication.