Origin licensing and p53 status regulate Cdk2 activity during G1

Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599, USA.
Cell cycle (Georgetown, Tex.) (Impact Factor: 4.57). 07/2009; 8(12):1952-63. DOI: 10.4161/cc.8.12.8811
Source: PubMed


Origins of DNA replication are licensed through the assembly of a chromatin-bound prereplication complex. Multiple regulatory mechanisms block new prereplication complex assembly after the G(1)/S transition to prevent rereplication. The strict inhibition of licensing after the G(1)/S transition means that all origins used in S phase must have been licensed in the preceding G(1). Nevertheless mechanisms that coordinate S phase entry with the completion of origin licensing are still poorly understood. We demonstrate that depletion of either of two essential licensing factors, Cdc6 or Cdt1, in normal human fibroblasts induces a G(1) arrest accompanied by inhibition of cyclin E/Cdk2 activity and hypophosphorylation of Rb. The Cdk2 inhibition is attributed to a reduction in the essential activating phosphorylation of T160 and an associated delay in Cdk2 nuclear accumulation. In contrast, licensing inhibition in the HeLa or U2OS cancer cell lines failed to regulate Cdk2 or Rb phosphorylation, and these cells died by apoptosis. Co-depletion of Cdc6 and p53 in normal cells restored Cdk2 activation and Rb phosphorylation, permitting them to enter S phase with a reduced rate of replication and also to accumulate markers of DNA damage. These results demonstrate dependence on origin licensing for multiple events required for G(1) progression, and suggest a mechanism to prevent premature S phase entry that functions in normal cells but not in p53-deficient cells.

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Available from: Jean Cook, Sep 07, 2015
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    • "Because of the inherent risk of trying to replicate the genome with too few licensed origins, it would make sense if cells had a way to ensure that a sufficient number of origins have been licensed in late G1 before the licensing system is shut down in preparation for entry into S phase. Consistent with this idea, it has been shown that certain metazoan cell lines possess a “licensing checkpoint” that prevents G1 cells from entering S phase before the licensing system is inhibited [49,51,56,69,74]. This checkpoint arrests cells in late G1 prior to full Cdk activation and consequent phosphorylation of the retinoblastoma protein Rb, at a cell cycle stage where further origin licensing should be possible. "
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    ABSTRACT: The ability of a eukaryotic cell to precisely and accurately replicate its DNA is crucial to maintain genome stability. Here we describe our current understanding of the process by which origins are licensed for DNA replication and review recent work suggesting that fork stalling has exerted a strong selective pressure on the positioning of licensed origins. In light of this, we discuss the complex and disparate phenotypes observed in mouse models and humans patients that arise due to defects in replication licensing proteins.
    Full-text · Article · Apr 2014 · DNA repair
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    • "Cip1 and p27 Kip1 (Shreeram et al. 2002; Machida et al. 2005; Teer et al. 2006; Liu et al. 2009). A second p53-dependent pathway involves loss of phosphorylation at Thr160 in the activation T loop of CDK2, accompanied by delayed nuclear accumulation of CDK2 protein in late G1 (Nevis et al. 2009). Depletion of p53 in primary cells abolished the licensing checkpoint, promoting Rb hyperphosphorylation and E2F transcriptional activity. "
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    ABSTRACT: Only ∼10% of replication origins that are licensed by loading minichromosome maintenance 2-7 (MCM2-7) complexes are normally used, with the majority remaining dormant. If replication fork progression is inhibited, nearby dormant origins initiate to ensure that all of the chromosomal DNA is replicated. At the same time, DNA damage-response kinases are activated, which preferentially suppress the assembly of new replication factories. This diverts initiation events away from completely new areas of the genome toward regions experiencing replicative stress. Mice hypomorphic for MCM2-7, which activate fewer dormant origins in response to replication inhibition, are cancer-prone and are genetically unstable. The licensing checkpoint delays entry into S phase if an insufficient number of origins have been licensed. In contrast, humans with Meier-Gorlin syndrome have mutations in pre-RC proteins and show defects in cell proliferation that may be a consequence of chronic activation of the licensing checkpoint.
    Full-text · Article · Aug 2012 · Cold Spring Harbor perspectives in biology
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    • "We also noted that knock-down of ORC2, Cdt1, Cdc6 or MCM7 as well as resulting in increased viral DNA replication resulted in robust increases in IE72 expression at IE times of infection - this could not be explained by, for instance, an increase in delivery of viral genome to cells lacking these cellular replication proteins. It is known that HCMV only initiates IE gene expression in cells in G0/G1 or very early S phase [54], [55], [56] and it is well established that depletion of ORC2, Cdt1, Cdc6 or MCM7 can result in cell cycle arrest at G0/G1 [40] [6], [57], [58], [59], [60], [61], [62]. Consequently, we tested whether cells treated with specific siRNAs to pre-RC factors under the conditions we used for our infection assays, resulted in major differences in the number of cells in G0/G1. "
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    ABSTRACT: Although HCMV encodes many genes required for the replication of its DNA genome, no HCMV-encoded orthologue of the origin binding protein, which has been identified in other herpesviruses, has been identified. This has led to speculation that HCMV may use other viral proteins or possibly cellular factors for the initiation of DNA synthesis. It is also unclear whether cellular replication factors are required for efficient replication of viral DNA during or after viral replication origin recognition. Consequently, we have asked whether cellular pre-replication (pre-RC) factors that are either initially associated with cellular origin of replication (e.g. ORC2), those which recruit other replication factors (e.g. Cdt1 or Cdc6) or those which are subsequently recruited (e.g. MCMs) play any role in the HCMV DNA replication. We show that whilst RNAi-mediated knock-down of these factors in the cell affects cellular DNA replication, as predicted, it results in concomitant increases in viral DNA replication. These data show that cellular factors which initiate cellular DNA synthesis are not required for the initiation of replication of viral DNA and suggest that inhibition of cellular DNA synthesis, in itself, fosters conditions which are conducive to viral DNA replication.
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