1996 Jan 11;379(6561):180-2. doi: 10.1038/379180a0. May recruit the MCM helicase onto the oriC region. Cdc6 is a key regulator of DNA replication in eukaryotes. Gopalakrishnan V, Simancek P, Houchens C, Snaith HA, Frattini MG, Sazer S, Kelly TJ. The expression of CDC6 at the end of mitosis suggested a role of the protein during G1 (20,21), which was confirmed using a ‘conditional knockout (KO)’ yeast strain. (A) Main conserved motifs in human CDC6 protein. In the case of non-small cell lung carcinomas, no direct correlation was observed between the levels of CDC6 and proliferation marker Ki67 (82). 1993 Dec;15(12):775-82. doi: 10.1002/bies.950151202. When hCDC6 levels were downregulated in G1 cells by antibody microinjection, cells could not progress into S phase (15,17). See text for details. (B) Schematic of the potential functions of CDC6 at different stages of the cell division cycle. The different conserved AAA+ boxes are indicated in yellow, except Walker A, Walker B, Sensor 1 and Sensor 2 that are indicated in green. Spanish Ministry of Education and Science (BFU2004-04886, CSD2007-00015); Marie Curie International Reintegration Grant from the European Union 6th Framework Programme (FP6-031129); Fundación Caja Madrid (CM-OM1108) to J.M. CDC6 oncogenic potential, clearly shown in tissue culture cells, ought to be confirmed in animal models. The human cell division cycle is more complex but the potential of human CDC6 to restrain mitosis seems conserved, at least when the protein is overexpressed in G2 cells (69). Furthermore, CDC6 overexpression increased colony formation and cooperated with oncogenic Ras to transform mouse embryo fibroblasts. DNA replication proteins serve as diagnosis markers of several neoplastic conditions and are the target of widely used antiviral and anticancer drugs. Because CDC6 and MCM proteins are normally absent in quiescent and differentiated cells, their immunohistochemical detection can be used for the early detection of malignancies. Exogenous glutathione alleviates chilling injury in postharvest bell pepper by modulating the ascorbate-glutathione (AsA-GSH) cycle. An interesting structural parallelism has been drawn between CDC6 and ‘clamp loaders’ such as replication factor C or the γ-complex of Escherichia coli DNA polymerase III (32), multisubunit complexes that use the energy derived from ATP hydrolysis to engage ring-shaped molecules such as proliferating cell nuclear antigen (PCNA) or the E.coli β-dimer with the DNA (45,46). The notion that aberrant DNA replication is a driving force of genomic instability is supported by several studies showing, both in yeast and mammalian cells, that the interference with pre-RC formation leads to inefficient S phase and increases aneuploidy and the frequency of gross chromosomal rearrangements (92–95). National Library of Medicine First, we discuss its biochemical roles in the activation of replication origins and the coordination of S phase and mitosis. Not all of them may apply to human CDC6. Early cellular response to oncogene activation. Centrosome number is tightly controlled during the cell cycle to ensure proper spindle assembly and cell division. It was quickly noted that ORC associated with other proteins in larger structures called pre-replication complexes (pre-RCs), which could be visualized by genomic footprinting at origins of replication during G1 (23). Then, in S phase, the Mcm2-7 hexamer becomes activated, leading to origin firing and DNA synthesis. INK4/ARF repression was mediated by the recruitment of histone deacetylases HDAC1 and HDAC2 that modified the epigenetic signature of the locus, leading to its eventual ‘heterochromatinization’ (89). In the early 1990s, a yeast origin of replication was used as a bait to isolate the six-subunit ‘origin recognition complex’ [ORC; (22)]. p53-independent targeted destruction of CDC6 has also been found after treatment of the cells with adozelesin (76), methyl methane sulfonate or UV irradiation (77). (2000) Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle. The levels of the corresponding proteins may be kept close to physiological levels by the action of their normal regulatory mechanisms, but in same cases they reach abnormally high concentrations. The answer can be learnt from the inspiring biography of Francis Crick (104), who ‘as a small boy was haunted by a fear that by the time he grew up everything would have been discovered’. In contrast, CDC6 deregulation does not lead to origin reactivation in Saccharomyces cerevisiae, due to overlapping mechanisms that involve the inactivation of additional initiator proteins (56). However, CDC6 also plays important roles in the activation and maintenance of the checkpoint mechanisms that coordinate S phase and mitosis, and recent studies have unveiled its proto-oncogenic activity. ORC is also an ATPase whose activity is not required for the loading of the first MCM complex but becomes essential for the repeated loading of additional MCMs. Functions of CDC6 in DNA replication and checkpoint activation. Overexpression of the related Cdc18 protein in fission yeast results in DNA rereplication; however, Cdc6p overexpression does not cause this result. One of them used a biochemical fractionation protocol to prove that MCM proteins could not associate with the chromatin in the absence of CDC6 (26). They published their findings in Molecular Cell. The deregulation of other DNA replication proteins has been linked to cancer in animal models. The main concept behind the control of DNA replication is the classic ‘replicon hypothesis’: specific DNA sequences serve as ‘origins of replication’ and are activated by soluble factors called ‘initiators’. Bioessays. These responses are elicited through the orchestrated action of multiple sensors, transducers or effectors. 1997 Oct 1;16(19):5988-97. doi: 10.1093/emboj/16.19.5988. The 50 kb INK4/ARF locus encodes three important tumor suppressor genes: p16INK4a and p15INK4b, both activators of the retinoblastoma pathway, and ARF, an activator of p53. EMBO J. CDC6 either directly (left) or indirectly (by binding a different DNA-bound protein; right) binds to DNA and recruits MCM2-7 and CDT1 to the origin of replication. In addition, DNA replication is carefully coordinated with cell division in order to maintain genomic integrity. Similarly, the loading of MCM proteins by CDC6 and ORC could be achieved by a series of conformational changes, coupled to ATP binding and hydrolysis, which open and close the MCM ring around the DNA. With these precedents, it will be very interesting to evaluate the effects of CDC6 overexpression in vivo (see ‘Questions for the future’ below). Cell division cycle 6 (CDC6) is an essential regulator of DNA replication in eukaryotic cells. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Cdc6p acts at replication origins, and cdc6‐1 mutants arrest with unreplicated DNA and show elevated minichromosome loss rates. The recent discovery of CDC6 oncogenic properties should prompt a systematic search for genetic alterations in CDC6 (gene amplification, loss or mutation) in a wide range of tumor samples. Later on, it was found that CDC6 downregulation by RNA interference (RNAi) prevented cell proliferation and promoted apoptosis (29,30). An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast. CDC6 is aberrantly overexpressed in most cancer types, and this not only promotes DNA replication and cell proliferation directly but may also facilitate oncogenesis as CDC6 can repress the transcription of E-cadherin and the tumor suppressor INK4/ARF locus However, the mechanisms that suppress CDC6 expression in normal cells and the cellular events that lead to CDC6 overexpression in … We thank Manuel Serrano for many stimulating discussions and Cecilia Sgarlata for the generation of a CDC6 transgenic mouse model. Pelizon,C., Madine,M.A., Romanowski,P. A comprehensive analysis of the posttranslational modifications of CDC6 and its stability in different cellular contexts may be needed to clarify this issue. Its best-characterized function is the assembly of prereplicative complexes at origins of replication during the G 1 phase of the cell division cycle. CDC6 stability relies on the phosphorylation of three N-terminal serine residues by CDKs, which protects it from ubiquitination and degradation (74,75). This interesting observation cleared the way to understand the participation of CDC6 in additional cellular functions, including checkpoint mechanisms and mitotic entry (see below). ATM–Chk2) to inhibit mitosis. In this review, we concentrate on the cellular functions and oncogenic properties of initiator protein cell division cycle 6 (CDC6). Therefore, in a situation of cellular stress, CDC6 is at the crossroads of two seemingly opposing pathways, one that stabilizes it in order to activate the S-M checkpoint signaling and another one that destabilizes it in order to avoid further initiation events (Figure 1B). CDT1 levels are controlled by proteolytic regulation mediated by SCFSkp2 or the Cul4-DDB1Cdt2 ubiquitin ligases. Inactivation of this locus is one of the most frequent events in human cancer (88). ORC and Cdc6 combine to form a ring-shaped complex that contains six AAA+ subunits. Additional diagnostic applications based on CDC6 and MCM immunodetection are being developed, including population screenings of bladder, colorectal, anal, lung and oral cancers (86). This cartoon includes CDC6 functions derived from different experimental systems. The process of genome replication that takes place in every mitotic cell cycle has always been considered a topic of ‘basic research’. An independent study revealed that a mutation in the ATP-binding site reduced the ability of CDC6 to cooperate with Cyclin E in the induction of DNA replication in quiescent, serum-starved cells. The nature of the signal sent by CDC6 to prevent premature entry into mitosis is independent from the origin-activating role, as a CDC6 mutant that did not support DNA replication still blocked mitosis (34). DNA replication Group, Molecular Oncology Programme, Spanish National Cancer Research Centre, Melchor Fernández Almagro 3, E-28029 Madrid, Spain. To test whether the MCM protein loaded by Cdc6-E224Q could promote DNA replication, that is, whether the dhMCM was functional, we expressed just Cdc6-E224Q (or Cdc6-WT) in a 2-hr window from M- to G1-phase and then monitored S-phase progression after switching off Cdc6 (mutant or WT) expression to allow Cdc6 removal. In the context of stalled replication forks caused by inhibitors of DNA replication, cells could stabilize CDC6 to induce the mitotic block, whereas in the context of extensive DNA damage cells would opt to degrade it to prevent origin activation, while relying on alternative pathways (e.g. DNA Replication and Human Disease, Control of DNA replication and its potential clinical exploitation, The regulation of INK4/ARF in cancer and aging, Oncogenic activity of Cdc6 through repression of the INK4/ARF locus, A new mechanism of inactivation of the INK4/ARF locus, Role of interactions between the origin recognition complex and SIR1 in transcriptional silencing, Deregulation of cyclin E in human cells interferes with prereplication complex assembly, The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G(1), Precocious G1/S transitions and genomic instability: the origin connection, Deregulated G1-cyclin expression induces genomic instability by preventing efficient pre-RC formation, Oncogene-induced senescence is a DNA damage response triggered by DNA hyper-replication, Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints, Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a, DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis, Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions, Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision, Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis, A viable allele of Mcm4 causes chromosome instability and mammary adenocarcinomas in mice, Francis Crick: Discoverer of the Genetic Code, © The Author 2007. Whether this ATR–interacting protein (ATRIP) ‘anchoring’ function is conserved in human CDC6 remains to be tested, but it is interesting to note that ATRIP interacts with MCM7 (73). DNA replication stress may be critical at the early stages of tumorigenesis (see Figure 2). A more trivial possibility is that different levels of CDC6 overexpression could tip the balance toward one outcome or the other. All rights reserved. Stillman and his colleagues have shown that CDC6 recruits other regulatory proteins that control the activity and levels of ORC1 in both space and time. The loading of several MCMs at a single origin is not surprising, as they are found on the chromatin in large excess relative to other initiator proteins (41).
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