Previous work has shown that the Myc transcription factor induces transcription of the E2F1, E2F2, and E2F3 genes. Using primary mouse embryo fibroblasts deleted for individual E2F genes, we now show that Myc-induced S phase and apoptosis requires distinct E2F activities. The ability of Myc to induce S phase is impaired in the absence of either E2F2 or E2F3 but not E2F1 or E2F4. In contrast, the ability of Myc to induce apoptosis is markedly reduced in cells deleted for E2F1 but not E2F2 or E2F3. From this data, we propose that the induction of specific E2F activities is an essential component in the Myc pathways that control cell proliferation and cell fate decisions.
XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junctioncutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNAbinding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.Nucleotide excision repair is a general repair system that plays an important role in maintaining genetic integrity (1, 2). This repair system removes damaged nucleotides from DNA by dual incisions on both sides of the lesion in the damaged strand (3). Recently, the human and the highly homologous yeast nucleotide excision repair systems have been reconstituted from extensively purified proteins, which demonstrated that 14 -15 polypeptides comprising replication protein A (RPA, 1 also known as HSSB or RF-A) and the general transcription factor, TFIIH, in addition to XP and ERCC proteins were essential for the dual incision step (4 -6).It was found that the two subunits of the excision nuclease had intrinsic endonuclease activities in the absence of the other components of the repair system. XPG was reported to possess a single-stranded DNA endonuclease activity (7,8) and an exonuclease activity with 5Ј to 3Ј directionality (9). Similarly, it was found that XPF-ERCC1 had a single strand-specific endonuclease activity and a weak activity on double-stranded UVirradiated DNA, which was stimulated by RPA (10). Furthermore, it has been reported that the RAD1-RAD10 complex (11), the yeast counterpart of the human XPF-ERCC1 nuclease, and XPG nuclease (12) have structure-specific junction-cutting activities with unique polarities. These results obtained with the model "undamaged" substrates suggested that XPF-ERCC1 and XPG were the 5Ј-and 3Ј-endonucleases of the dual incisions, respectively. A recent study using damaged DNA and specific antibodies supported this model (13).Recently, it has been reported that RPA, which is absolutely required for dual incisions (4), specifically binds to XPA protein and XPG nuclease (14). These findings raised the possibility that RPA may play an important role in recognizing DNA lesions and then targeting the nuclease subunits of hu...
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...
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