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Initiation of DNA replication in eukaryotic cells is regulated by an `origin licensing' mechanism that requires the sequential assembly of proteins into pre-replicative complexes (pre-RCs) at origins of replication, the sites on chromosomes where DNA synthesis begins (reviewed in reference). Activation of origins in S-phase by cyclin-dependent kinases (CDKs) coincides with the inactivation and partial disassembly of pre-RCs, and CDKs and other proteins block their reassembly until the end of mitosis, when CDK activity is destroyed (Figure 1, top panel). The reciprocal relationship between pre-RCs and CDK activity provides a stringent, failsafe mechanism for ensuring that DNA is replicated just once per cell cycle. Although the details are sometimes different, this basic mechanism for regulating chromosome duplication appears to be conserved in all eukaryotes. Recent experiments suggest the hypothesis that, in addition to regulating DNA replication, origin licensing plays an important role in regulating cell death. They show that multiple p53-independent, proteasome and/or caspasedependent pathways destroy the replication initiation protein Cdc6, which is required for the assembly and/or maintenance of pre-RCs, at an early stage of apoptosis. The proteasome-dependent destruction of Cdc6, which was induced by the DNA-damaging drug adozelesin, occurs upstream of, or parallel to, the action of caspases. This pathway is conserved in budding yeast, where mutations in pre-RC proteins that interact with Cdc6 had previously been shown to alter the sensitivity of budding yeast cells to this drug. In mammalian cells, the caspase-dependent destruction of Cdc6 was mediated by an extrinsic apoptotic pathway induced by TNF-a. How might the destruction of Cdc6 contribute to cell death? In yeast and mammals, Cdc6 and some other proteins in pre-RCs are absent from quiescent cells, and licensing complexes are assembled upon entry into the cell cycle. In mammals, this occurs when proliferative signals activate the expression of licensing proteins regulated by the E2F family of transcription factors, downstream of cyclin D1 synthesis and the subsequent phosphorylation of members of the retinoblastoma protein (Rb) family. Thus, pre-RC assembly occurs at or just beyond the point where many of the pathways that regulate proliferation converge. In fact, these pathways may physically converge on origins of replication. In yeast, ± 7 Xenopus and humans, for instance, pre-RC proteins, including Cdc6, have been shown to interact with, and in some cases inhibit, CDKs required for progression through mitosis. Recent studies in mammals and Drosophila have also detected physical associations between components of pre-RCs and Rb and/ or E2F, both of which have been implicated in DNA damage responses and apoptosis in addition to their roles in regulating proliferation. In Drosophila, a component of pre-RCs also regulates chromosome condensation and cohesion in preparation for mitosis. All of these findings suggest that in eukaryotic cells, Cdc6 and other components of pre-RCs form a nexus for cell cycle regulation where DNA replication is coordinated with mitosis downstream of signaling pathways that regulate cell proliferation, and perhaps DNA damage responses and cell death. One might predict, therefore, that loss of integrity of preRCs during apoptosis in proliferating cells would result in aberrant regulation of the cell cycle. In fact, accumulating evidence suggests that disregulation of the cell cycle is an important component of mammalian cell death pathways. At the simplest level, there is the longstanding observation that cycling cells are more sensitive to a variety of apoptotic triggers compared to quiescent cells. A clear in vivo example of this relationship is provided by studies of epithelial cells in the small intestine of mice exposed to ionizing radiation and other cytotoxic agents. While cells in the proliferative compartment of this tissue are sensitive to these apoptosis inducers, they become refractile once they undergo a defined transition to quiescence. The sensitization of proliferating cells to apoptosis induced by some apoptotic triggers can be duplicated by the forced overexpression of a number of oncoproteins in quiescent cells. Some studies have established direct connections between elevated levels of oncoproteins and the activity of CDKs during apoptosis. In fact, a compelling argument can now be made that the activation of CDKs is, in many cases, a requisite feature of programmed cell death in mammals (reference and references therein). Perhaps the strongest argument may be the observation that many aspects of apoptosis are inhibited when CDK activity is blocked by dominant-negative CDK mutants. This has now been observed in a variety of systems and in association with a number of different apoptotic triggers, including those required for normal differentiation processes. Interestingly, in yeast (reference and references therein) and Xenopus, the elimination of Cdc6 or the failure to form pre-RCs in G1 cells results in a CDK-dependent reductional anaphase when cells pass through mitosis in the absence of DNA replication. Recent experiments in budding yeast suggest this is due to the loss of a mitotic checkpoint that at least partly depends on inhibitory Cell Death and Differentiation (2002) 9, 870 ± 872 a 2002 Nature Publishing Group All rights reserved 1350-9047/02 $25.00 |
