Genetic screening of yeast for sld (synthetic lethality with dpb11) mutations has identified replication proteins, including Sld2, -3, and -5, and clarified the molecular mechanisms underlying eukaryotic chromosomal DNA replication. Here, we report a new replication protein, Sld7, identified by rescreening of sld mutations. Throughout the cell cycle, Sld7 forms a complex with Sld3, which associates with replication origins in a complex with Cdc45, binds to Dpb11 when phosphorylated by cyclin-dependent kinase, and dissociates from origins once DNA replication starts. However, Sld7 does not move with the replication fork. Sld7 binds to the nonessential N-terminal portion of Sld3 and reduces its affinity for Cdc45, a component of the replication fork. Although Sld7 is not essential for cell growth, its absence reduces the level of cellular Sld3, delays the dissociation from origins of GINS, a component of the replication fork, and slows S-phase progression. These results suggest that Sld7 is required for the proper function of Sld3 at the initiation of DNA replication.
c Dpb11/Cut5/TopBP1 is evolutionarily conserved and is essential for the initiation of DNA replication in eukaryotes. The Dpb11 of the budding yeast Saccharomyces cerevisiae has four BRCT domains (BRCT1 to -4). The N-terminal pair (BRCT1 and -2) and the C-terminal pair (BRCT3 and -4) bind to cyclin-dependent kinase (CDK)-phosphorylated Sld3 and Sld2, respectively. These phosphorylation-dependent interactions trigger the initiation of DNA replication. BRCT1 and -2 and BRCT3 and -4 of Dpb11 are separated by a short stretch of ϳ100 amino acids. It is unknown whether this inter-BRCT region functions in DNA replication. Here, we showed that the inter-BRCT region is a GINS interaction domain that is essential for cell growth and that mutations in this domain cause replication defects in budding yeast. We found the corresponding region in the vertebrate ortholog, TopBP1, and showed that the corresponding region also interacts with GINS and is required for efficient DNA replication. We propose that the inter-BRCT region of Dpb11 is a functionally conserved GINS interaction domain that is important for the initiation of DNA replication in eukaryotes. Chromosomal DNA replication in eukaryotes occurs as a twostep reaction whose steps are separated temporally in the cell cycle (see reviews in references 1 and 2). In the first reaction, called licensing, the core component of the replicative helicase, Mcm2-7, is loaded onto replication origins to assemble the prereplicative complexes (pre-RCs) in the G 1 phase of the cell cycle. At this point, the Mcm2-7 helicase is inactive in the pre-RC. In the following S phase, the replicative helicase is activated by the functions of two protein kinases-cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK)-and the replication fork is established (initiation reaction). In the budding yeast Saccharomyces cerevisiae, Sld2 and Sld3 are essential targets of CDK. CDK phosphorylation of Sld2 and Sld3 promotes a phosphodependent interaction with the tandem BRCT repeats of another replication factor called Dpb11 (3-6). These interactions promote the recruitment of GINS onto origins via the preloading complex (pre-LC), which consists of Dpb11, Sld2, DNA polymerase ε (Pol ε), and GINS. The pre-LC is an important intermediate in the subsequent formation of the active replicative helicase, which is called the CMG (Cdc45-Mcm2-7-GINS) complex, and the establishment of the replication fork (7-9). Therefore, Dpb11 is a key mediator of the formation of the new complex on replication origins and plays a crucial role in the initiation of DNA replication.Dpb11 is evolutionarily conserved (10, 11). The functions and overall structures of Dpb11 of budding yeast and of its ortholog in fission yeast, Cut5, are similar (12, 13) (see Fig. S1 in the supplemental material). The vertebrate ortholog, TopBP1, is also a BRCT-containing protein (14), the N-terminal region of which contains BRCT1 and -2, which interacts with phosphorylated treslin, an ortholog of vertebrate Sld3 (15,16). Although TopBP1 is also c...
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