Abstract
Treslin/Ticrr is required for the initiation of DNA replication and binds to MTBP (Mdm2 Binding Protein). Here, we show that in Xenopus egg extract, MTBP forms an elongated tetramer with Treslin containing two molecules of each protein. Immunodepletion and add-back experiments show that Treslin-MTBP is rate limiting for replication initiation. It is recruited onto chromatin before S phase starts and recruitment continues during S phase. We show that DDK activity both increases and strengthens the interaction of Treslin-MTBP with licensed chromatin. We also show that DDK activity cooperates with CDK activity to drive the interaction of Treslin-MTBP with TopBP1 which is a regulated crucial step in pre-initiation complex formation. These results suggest how DDK works together with CDKs to regulate Treslin-MTBP and plays a crucial in selecting which origins will undergo initiation.
Highlights
The initiation of eukaryotic DNA replication occurs at replication origins that have been licensed by being loaded with double hexamers of MCM2–7 proteins
We show that in Xenopus egg extracts, MTBP and Treslin form an elongated complex that is recruited to DNA before replication initiates
The Treslin–MTBP complex can be recruited to DNA in a functional form in the absence of replication licensing, DDK or CDK activity, but licensing and DDK activity stabilizes the interaction between Treslin– MTBP and chromatin-bound MCM2-7
Summary
The initiation of eukaryotic DNA replication occurs at replication origins that have been licensed by being loaded with double hexamers of MCM2–7 proteins. The combined action of two kinases, DDK and S-CDK, triggers the recruitment of Cdc and the GINS complex onto these MCM2-7 double hexamers to form the replicative CMG (Cdc45–MCM–GINS) helicase. In Saccharomyces cerevisiae, DDK phosphorylation of MCM2–7 promotes the recruitment of Sld and Cdc to origins, and S-CDK phosphorylation of Sld and Sld allows the recruitment of Dbp, Sld, Sld, GINS and Pol ε [5,6,7,8]. DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 targeted to chromatin by Rif in Xenopus egg extract and in both S. cerevisiae and human cells [3,4,9,10]
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