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

Abstract

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 the S phase of the budding yeast cell cycle. However, the molecular basis for such temporal regulation is poorly understood. We show that origin association of the low-abundance 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. The DDK-dependent limited association between origins and Sld3-Sld7-Cdc45 is a key step for determining the timing of origin firing.