Analysis of Cdc6 function in the assembly of mammalian prereplication complexes

Abstract

T he initiation of DNA replication in eukaryotic cells requires the assembly of multiple proteins at origins together with the action of protein kinases. Among the proteins known to assemble at origins before the initiation of DNA synthesis are the origin recognition complex, Cdc6, and the minichromosome maintenance (Mcm) complex. Together, these constitute a ''prereplication'' complex to which additional factors bind once cyclin-dependent kinases (cdks) become active. Once all of the necessary proteins have assembled at the origin, additional phosphorylation events trigger initiation (for reviews see refs. 1-5).Mutational alteration in yeast (6-8) or immunodepletion of Xenopus laevis oocytes (9) has demonstrated that Cdc6 and each origin recognition complex and MCM subunit plays a unique and essential role in replication. Cdc6 is thought to act primarily through recruitment of the Mcm complex to origins because mutations in yeast cdc6 or immunodepletion of X. laevis Cdc6 lead to a loss of Mcm origin association (9-14). Although homologs of virtually all of the proteins implicated in DNA replication initiation have been identified in mammals, the analysis of these counterparts has been hampered by the lack of in vivo experimental systems. In addition, many of the regulatory mechanisms that target replication factors may not be strictly conserved across species. For instance, phosphorylation of Saccharomyces cerevisiae Cdc6 (and the corresponding Schizosaccharomyces pombe Cdc18 protein) by cdks leads to proteolytic degradation (15-18), whereas in X. laevis oocyte extracts and in mammalian cells, cdk phosphorylation of Cdc6 leads to export of the protein from the nucleus to the cytoplasm (19-21). Furthermore, the peak expression time of the yeast Cdc6 homologs appears to be early G 1 whereas human Cdc6 is specifically degraded in G 1 and accumulates to highest levels in G 2 ͞M (22, 23). For these reasons, it is clearly important to extend the investigation of the function and regulation of mammalian Cdc6 in its native setting. We have made use of recombinant adenovirus vectors to examine Cdc6 function in intact quiescent mammalian cells. Adenovirus-mediated expression is a particularly powerful tool because of the ability of the virus to infect an entire population of cells under both growing and quiescent conditions. Using this approach, we provide evidence that Cdc6 is not only necessary for Mcm chromatin association but is sufficient to induce endogenous Mcm chromatin loading in serum-deprived cells. We also show that Cdc6 synergizes with limiting amounts of cyclin E͞Cdk2 to induce semiconservative DNA replication, and we use this system to begin to identify domains of Cdc6 critical for this function. Materials and MethodsCells and Viruses. REF52 cells were grown in DMEM and brought to quiescence as described (24). Infections were carried out in DMEM plus 25 mM Hepes, pH 7.5 for 75 min at 37°C in 20 l͞cm 2 . Adenoviral stocks were purified and maintained as described (25). The Ad-CycE, Ad-Cdk2 (containing a hem...

E2F2 represses cell cycle regulators to maintain quiescence

“…Inspection of these clusters revealed that the E2F regulated genes are involved in a wide spectrum of pathways. To determine whether gene enrichment in a given functional category was statistically significant (relative to the representation of genes within that functional category in our cDNA microarray) we used the EASE algorithm, 31 considering separately the two time points (quiescence and G 1 /S) and up versus 33 Given the fact that quiescent DKO cells exhibit increased levels of Cdc6 and Mcm protein we asked whether the Mcm proteins are associated with chromatin in these cells even during quiescence. Cells were lysed and fractionated into a chromatin enriched pellet and a soluble supernatant.…”

CDK1-dependent Inhibition of the E3 Ubiquitin Ligase CRL4CDT2 Ensures Robust Transition from S Phase to Mitosis

“…Whole-cell lysates were prepared either by direct lysis of equal cell numbers in 2ϫ Laemmli sample buffer with 10% ␤-mercaptoethanol or in CSK buffer (supplemented with 0.1% Triton X-100 and protease and phosphatase inhibitors) (34). Chromatin-enriched fractions were isolated as described previously (35) by the addition of micrococcal nuclease and CaCl 2 to Triton-insoluble pellets to release DNA-bound material into the soluble pool and clarified by centrifugation. Alternatively, sonication was used to solubilize the chromatin-bound proteins.…”

Quantitation of CDC6 and MCM5 mRNA in cervical intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix

“…Among the proteins known to assemble to form the pre-RC are cell division cycle protein 6 (CDC6) and mini chromosome maintenance (MCM) proteins. 3,4 Biological analysis of CDC6 suggests that it functions as a clamp loader in which ATP binding and hydrolysis induce conformational changes, which result in the recruitment or loading of MCMs onto DNA. 3 Once the MCMs are recruited to the pre-RC, CDC6 protein is then phosphorylated by Cyclin A/CDK2 in S phase of the cell cycle.…”