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The initiation of eukaryotic DNA replication occurs at replication origins, licensed by loaded double hexamers of Mcm2-7 proteins. The action of two kinases, DDK and S-CDK, triggers the loading onto Mcm2-7 of Cdc45 and the GINS complex to form the replicative CMG helicase. In yeast, the proteins Sld2 and Sld3 are CDK substrates that interact with Dpb11, and are required for the assembly of the CMG complex. Sld7 has been reported to associate with Sld3 throughout the cell cycle and regulate the function of Sld3 at replication initiation. Metazoan orthologues of these proteins have been identified: TopBP1, for Dpb11, is essential for replication;; Treslin, the Sld3 ortholog, is an essential S-CDK substrate that interacts with TopBP1 and is required for the CMG assembly;; in human cells MTBP has been found associated with Treslin, and is involved in the regulation of DNA replication, therefore MTBP as been proposed as a potential Sld7 ortholog. The aim of this project was to determine the role of MTBP in DNA replication using the Xenopus egg extract cell-free system. I found that MTBP and Treslin co-immunoprecipitate in metaphase and interphase extracts, suggesting that they form a complex throughout the cell cycle. MTBP, like Treslin, is recruited onto chromatin before the beginning of DNA replication and during S phase, independently of licensing and CDK/DDK activity. Immunodepletion of MTBP from extract leads to co- depletion of Treslin and causes a strong inhibition of DNA replication and impairment of CMG assembly. A partially purified fraction of extract, enriched for MTBP and Treslin, can rescue DNA replication in extracts depleted of MTBP. Analyses of the stoichiometry of the MTBP-Treslin complex has been carried out using gel filtration and sucrose gradient centrifugation; these identify a complex with an apparent molecular weight close to the one expected for a tetramer composed by two molecules of MTBP and two molecules of Treslin, resembling the model proposed for the interaction of the yeast proteins Sld3 and Sld7. These results, taken together, support the hypothesis that MTBP and Treslin form a multimeric complex that is required for the initiation of DNA replication and suggests that MTBP could be required to correctly position two Treslin molecules onto the MCM double hexamer to allow the bi-orientated firing of replication origins.