A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication

We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation.We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.The eukaryote genome is organised into multiple chromosomes, whose replication must be strictly controlled in order to ensure that the DNA is replicated once and only once in each cell cycle. DNA replication initiates from multiple origins, whose activation can be divided into two stages. In the first stage, pre-RCs are assembled at replication origins by the sequential binding of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 (the MCM/P1 proteins) [1,2]. This assembly takes places during late mitosis and G1, and results in the origin becoming "licensed" for DNA replication. The second stage occurs during S phase and involves the activation of licensed origins by the action of two S phase-promoting kinases: S-phase promoting CDKs and Cdc7/Dbf4, leading to the loading of Cdc45 and the initiation of a pair of replication forks.Cdc7 is a serine/threonine kinase, conserved from yeast to humans that is required fo