The initiation of DNA replication can be divided into three subsequent steps. First, a pre-replicative complex (pre-RC) is assembled that consists of the origin recognition complex (ORC), Cdc6, and Cdt1 as well as the minichromosome maintenance (Mcm)2-7 proteins. Secondly, the pre-RC is converted into the initiation complex (IC), which requires S-phase kinases for the timely "firing" of each individual origin of replication. Lastly, the initiation complex governs DNA unwinding at the origin, and the loading of the replicative DNA polymerases α and ε. The initiation factors Cdc45 and TopBP1 are crucial for the conversion of the pre-RC into the IC and have been discussed to be loading factors for DNA polymerases.
Recently, we have set up assay systems to determine physical and functional interactions between Cdc45p and the DNA polymerases α, δ and ε. Cdc45p was shown to be absent from quiescent cells and to become induced when cells reenter the cell cycle. Cdc45p is loaded into chromatin when cells enter S phase, where it co-localizes with the presumptive replicative DNA helicase Mcm2-7, and the DNA polymerases, probably as part of the ongoing replication fork. This suggests that Cdc45p acts, maybe together with the four-subunits of the GINS complex, as a bridging factor between the DNA polymerases and the Mcm2-7 helicase.
In collaboration with the Hänel group (Hans-Knöll-Institute Jena) we could show that Cdc45p binds to TopBP1, a multifunctional protein involved in replication, transcription, DNA repair, and the checkpoint-control of the cell cycle. Interestingly, TopBP1 also seems to bind to the RecQL4 protein, another DNA unwinding enzyme that is involved in the initiation of DNA replication. Presently, we are postulating the existence of a (transient) "TRC complex" consisting of TopBP1, ReqQL4 and Cdc45p as a functional module for the initiation of DNA replication that fulfills similar roles as the Sld2 and Sld3 proteins in budding yeast. Once this module becomes operative by the S-phase kinases, Cdc45p may load the replicative DNA polymerases into the activated origins and thereby constitute the mobile replicative machinery, where the polymerases together with Cdc45p and the Mcm2-7 helicase leave the origin, while TopBP1 and possible also RecQL4 are no longer part of the migrating fork.
Currently, we are using several methodologies to proof or disproof our working hypothesis. We have adapted a human nuclei-based in vitro replication system, which allows us to study the role of the studied proteins e.g. by inhibition, depletion and reconstitution of replication factors at will. This should enable us to reconstitute in vitro replication with purified fragments of the replication factors, such as the N- or C-terminal parts of Cdc45p, TopBP1 or RecQL4.
The TRC complex as a hypothetical replication initiation module
This project is part of the  and entitled as project B2 "Regulation of Eukaryotic DNA Replication".
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