Molecular mechanism of assembly of replication proteins at replication origins in eukaryotes

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At replication origins in eukaryotic cells, the pre-Replicative Complex (pre-RC) forms from late M phase to G1 phase when CDK activity is low. When CDK is activated at late G1 phase, many replication proteins assemble on the pre-RC to initiate DNA replication. However, how they assemble at origins had not been elucidated. This is because CDK substrates and CDK-dependent reaction at the initiation step of DNA replication was not known. In this study, we first identified Sld2 and Sld3 as CDK substrates essential for initiation of DNA replication. Both CDK-phosphorylated Sld2 and Sld3 bind to Dpbll. These bindings are essential for initiation of DNA replication, so that when both binding are bypassed DNA replication initiates in the absence of CDK activity. Sld2 has 11 CDK phosphorylation motifs. Phosphorylation of Thr84 at one of these motifs is a single determinant for binding to Dpbll while other Sld2 phosphorylations are prerequisites for Thr84 phosphorylation. This regulation seems to contribute to fine-tuning of origin firing. Sld3 forms a complex with a novel factor, Sld7 and functions for initiation of DNA replication. Next, we found formation of the pre-Loading Complex (pre-LC) as a CDK-dependent reaction. The pre-LC is a novel complex containing DNA polymerase ε, GINS, Sld2 and Dpbll, which formation depends on CDK activity but not association with origins or the pre-RC. We thus propose the model; when Sld2 is phosphorylated by CDK and binds to Dpbll the pre-LC forms; since Sld3 associates with origins in G1 phase it is phosphorylated by CDK at origins; the phosphorylated Sld3 recruits the pre-LC to origins through Dpbll; then, DNA replication initiates.