Little is known about the architecture and biochemical composition of the eukaryotic DNA replication fork. To study this problem, we used biotin-streptavidin-modified plasmids to induce sequence-specific replication fork pausing in Xenopus egg extracts. Chromatin immunoprecipitation was employed to identify factors associated with the paused fork. This approach identifies DNA pol alpha, DNA pol delta, DNA pol epsilon, MCM2-7, Cdc45, GINS, and Mcm10 as components of the vertebrate replisome. In the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45, GINS, and MCM2-7 are enriched at the pause site. The data suggest the existence of a large molecular machine, the "unwindosome," which separates DNA strands at the replication fork and contains Cdc45, GINS, and the MCM2-7 holocomplex.

Replication licensing factor is thought to be involved in the strict control of the initiation of DNA replication in eukaryotes. We identified a 100 kDa protein as a candidate for the licensing factor in Xenopus egg extracts. This protein was required for replication; it bound to sperm DNA before the formation of nuclei and apparently dissociated from the nuclear DNA during the progression of replication without being transported into the nuclei. An immunologically homologous protein in HeLa cells behaved similarly to the Xenopus protein during the cell cycle. Cloning and sequencing of the cDNAs encoding the Xenopus and human proteins revealed that they are homologs of yeast Mcm3, a putative yeast DNA replication licensing factor.

Recruitment of DNA polymerases onto replication origins is a crucial step in the assembly of eukaryotic replication machinery. A previous study in budding yeast suggests that Dpb11 controls the recruitment of DNA polymerases ␣ and onto the origins. Sld2 is an essential replication protein that interacts with Dpb11, but no metazoan homolog has yet been identified. We isolated Xenopus RecQ4 as a candidate Sld2 homolog. RecQ4 is a member of the metazoan RecQ helicase family, and its N-terminal region shows sequence similarity with Sld2. In Xenopus egg extracts, RecQ4 is essential for the initiation of DNA replication, in particular for chromatin binding of DNA polymerase ␣. An N-terminal fragment of RecQ4 devoid of the helicase domain could rescue the replication activity of RecQ4-depleted extracts, and antibody against the fragment inhibited DNA replication and chromatin binding of the polymerase. Further, N-terminal fragments of RecQ4 physically interacted with Cut5, a Xenopus homolog of Dpb11, and their ability to bind to Cut5 closely correlated with their ability to rescue the replication activity of the depleted extracts. Our data suggest that RecQ4 performs an essential role in the assembly of replication machinery through interaction with Cut5 in vertebrates.

We have identified Xenopus homologs of the budding yeast Sld5 and its three interacting proteins. These form a novel complex essential for the initiation of DNA replication in Xenopus egg extracts. The complex binds to chromatin in a manner dependent on replication licensing and S-phase CDK. The chromatin binding of the complex and that of Cdc45 are mutually dependent and both bindings require Xenopus Cut5, the yeast homolog of which interacts with Sld5. On replicating chromatin the complex interacts with Cdc45 and MCM, putative components of replication machinery. Electron microscopy further reveals that the complex has a ring-like structure. These results suggest that the complex plays an essential role in the elongation stage of DNA replication as well as the initiation stage.

Cyclins A and E and their partner cyclin-dependent kinases (Cdks) are key regulators of DNA synthesis and of mitosis. Immunofluorescence studies have shown that both cyclins are nuclear and that a proportion of cyclin A is localized to sites of DNA replication. However, recently, both cyclin A and cyclin E have been implicated as regulators of centrosome replication, and it is unclear when and where these cyclin-Cdks can interact with cytoplasmic substrates. We have used live cell imaging to study the behavior of cyclin/Cdk complexes. We found that cyclin A and cyclin E are able to regulate both nuclear and cytoplasmic events because they both shuttle between the nucleus and the cytoplasm. However, we found that there are marked differences in their shuttling behavior, which raises the possibility that cyclin/Cdk function could be regulated at the level of nuclear import and export. In the course of these experiments, we have also found that, contrary to published results, mutations in the hydrophobic patch of cyclin A do affect Cdk binding and nuclear import. This has implications for the role of the hydrophobic patch as a substrate selection motif. INTRODUCTIONSuccessive waves of cyclin-dependent kinase (Cdk) activity control progress through the eukaryotic cell cycle. Cdks are activated by binding a member of the cyclin family and phosphorylation by Cdk-activating kinase. The cyclin-Cdks that have been most strongly implicated in controlling entry into, and progress through, DNA replication are cyclins A and E. Both cyclins bind to Cdk2 (Elledge and Spottswood, 1991;Tsai et al., 1991;Koff et al., 1992), and the levels of both cyclins are strictly regulated throughout the cell cycle, by transcriptional and proteolytic mechanisms. Cyclin E levels are primarily dictated by the rate of its transcription because it is an unstable protein that is rapidly degraded by two different pathways of ubiquitin-dependent proteolysis (Clurman et al., 1996;Won and Reed, 1996;Singer et al., 1999;Wang et al., 1999;Winston et al., 1999;Nakayama et al., 2000). In contrast, cyclin A is stable until cells enter mitosis. Cyclin A levels first start to increase at the beginning of S phase and continue to rise throughout S and G2 phases until prometaphase, when they are rapidly degraded by ubiquitin-dependent proteolysis (Pines and Hunter, 1990;Hunt et al., 1992).There is extensive evidence to indicate that the activation of cyclin E/Cdk2 leads to the initiation of DNA replication. Cyclin E/Cdk2 activity is maximal at G1/S (Koff et al., 1992), replication of DNA in vitro is dependent on cyclin E/Cdk2 activity (Jackson et al., 1995;Strausfeld et al., 1996;Krude et al., 1997) and, in vivo, cyclin E is essential for DNA replication in Drosophila (Sauer et al., 1995). Cyclin A/Cdk2 has also been demonstrated to promote DNA replication (Girard et al., 1991;Pagano et al., 1992;Zindy et al., 1992;Strausfeld et al., 1996). However, cyclin A may be more significant in regulating progression through S phase (Connell-Crowley et al., 1998), perhap...