The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC Cdh1 ) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC Cdh1 ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage. INTRODUCTIONDuplication of large mammalian genomes requires that DNA replication initiate at thousands of chromosomal origins. In order for an origin to be competent for replication, it must first be bound by a multiprotein complex, the prereplication complex (preRC). PreRCs are constructed in a stepwise process through the chromatin binding of the origin recognition complex (ORC), which then recruits both the Cdc6 ATPase and Cdt1, two proteins that are required for the stable loading of the minichromosome maintenance complex (MCM). The Cdc6 and Cdt1-dependent loading of MCM complexes at origins licenses them for replication during the G1 phase of the cell cycle. Sufficient preRCs must be assembled during G1 to promote complete replication, but new preRCs must not assemble after S phase begins because relicensing of previously fired origins leads to rereplication and genome instability (Vaziri et al., 2003;Melixetian et al., 2004;Zhu et al., 2004;Archambault et al., 2005). For these reasons, preRC assembly is one of the most highly regulated events in the control of DNA replication. Cells restrict preRC assembly to the G1 period through a combination of overlapping mechanisms that regulate individual preRC components (reviewed in Bell and Dutta, 2002;Blow and Hodgson, 2002;Nishitani and Lygerou, 2002;Diffley, 2004;Machida et al., 2005;Fujita, 2006).Cdc6 is not only an essential factor for preRC construction, but it has also been implicated in the activation of the cell cycle checkpoint that prevents entry into mitosis while DNA replication is incomplete (Clay-Farrace et al., 2003;Oehlmann et al., 2004;Lau et al., 2006). These observations suggest that Cdc6 functions not only during G1, but also in later cell cycle stages. Moreover, Cdc6 plays a role in setting the threshold for...

DNA replication is tightly coordinated both with cell cycle cues and with responses to extracellular signals to maintain genome stability. We discovered that human Cdt1, an essential origin licensing protein whose activity must be restricted to G 1 phase, is a substrate of the stress-activated mitogen-activated protein (MAP) kinases p38 and c-Jun N-terminal kinase (JNK). These MAP kinases phosphorylate Cdt1 both during unperturbed G 2 phase and during an acute stress response. Phosphorylation renders Cdt1 resistant to ubiquitinmediated degradation during S phase and after DNA damage by blocking Cdt1 binding to the Cul4 adaptor, Cdt2. Mutations that block normal cell cycle-regulated MAP kinase-mediated phosphorylation interfere with rapid Cdt1 reaccumulation at the end of S phase. Phosphomimetic mutations recapitulate the stabilizing effects of Cdt1 phosphorylation but also reduce the ability of Cdt1 to support origin licensing. Two other CRL4 Cdt2 targets, the cyclin-dependent kinase (CDK) inhibitor p21 and the methyltransferase PR-Set7/Set8, are similarly stabilized by MAP kinase activity. These findings support a model in which MAP kinase activity in G 2 promotes reaccumulation of a low-activity Cdt1 isoform after replication is complete.Precise and complete genome duplication presents a unique challenge during the cell division cycle. To permit efficient replication, DNA synthesis initiates at many chromosomal sites, known as origins of DNA replication. During G 1 phase, origins are loaded with an inactive form of the DNA helicase core, the minichromosome maintenance (MCM) complex. Origins with loaded MCM complexes are "licensed" because they are competent for replication initiation in the subsequent S phase. MCM loading is accomplished through recruitment of MCM complexes from the nucleoplasm by the Cdt1 protein to an origin-bound assembly of the origin recognition complex (ORC) and the Cdc6 protein. ORC and Cdc6 then load MCM onto DNA (48,57,58).Failure to properly control MCM loading can lead to replication errors and genome instability if insufficient origin licensing occurs in G 1 or if inappropriate origin relicensing occurs after the onset of S phase. For example, high levels of Cdt1 or Cdc6 activity in S or G 2 phase can promote origin relicensing, which leads to extensive rereplication and cell death; modest deregulation of either Cdc6 or Cdt1 promotes genome instability and tumorigenesis (5,28,44). Thus far, the best-understood mechanisms restricting origin licensing to G 1 phase are cell cycle-regulated accumulation and degradation of licensing proteins and inhibition of several licensing proteins after Sphase onset through phosphorylation by cyclin-dependent kinases (CDKs) (7,23,34).Given the critical need to maintain tight control and coordination of origin licensing, it is likely that additional important regulatory mechanisms have yet to be uncovered. Cell cycle progression is arrested in response to a variety of cellular stresses, including exposure to inflammatory cytokines, bacterial toxins,...

LincRNA-p21 is a long noncoding RNA and a transcriptional target of p53 and HIF-1α. LincRNA-p21 regulates gene expression in cis and trans, mRNA translation, protein stability, the Warburg effect, and p53-dependent apoptosis and cell cycle arrest in doxorubicin-treated mouse embryo fibroblasts. p53 plays a key role in the response of skin keratinocytes to UVB-induced DNA damage by inducing cell cycle arrest and apoptosis. In skin cancer development, UVB-induced mutation of p53 allows keratinocytes upon successive UVB exposures to evade apoptosis and cell cycle arrest. We hypothesized that lincRNA-p21 has a key functional role in UVB-induced apoptosis and/or cell cycle arrest in keratinocytes and loss of lincRNA-p21 function results in the evasion of apoptosis and/or cell cycle arrest. We observed that lincRNA-p21 transcripts are highly inducible by UVB in mouse and human keratinocytes in culture and in mouse skin in vivo. LincRNA-p21 is regulated at the transcriptional level in response to UVB, and the UVB induction of lincRNA-p21 in keratinocytes and in vivo in mouse epidermis is primarily through a p53-dependent pathway. Knockdown of lincRNA-p21 blocked UVB-induced apoptosis in mouse and human keratinocytes, and lincRNA-p21 was responsible for the majority of UVB-induced and p53-mediated apoptosis in keratinocytes. Knockdown of lincRNA-p21 had no effect on cell proliferation in untreated or UVB-treated keratinocytes. An early event in skin cancer is the mutation of a single p53 allele. We observed that a mutant p53+/R172H allele expressed in mouse epidermis (K5Cre+/tg;LSLp53+/R172H) showed a significant dominant-negative inhibitory effect on UVB-induced lincRNA-p21 transcription and apoptosis in epidermis. We conclude lincRNA-p21 is highly inducible by UVB and has a key role in triggering UVB-induced apoptotic death. We propose that the mutation of a single p53 allele provides a pro-oncogenic function early in skin cancer development through a dominant inhibitory effect on UVB-induced lincRNA-p21 expression and the subsequent evasion of UVB-induced apoptosis.

The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that metazoan origins initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion or overexpression of Cdt1 or Cdc6 in human cells causes rereplication, a form of endogenous DNA damage. Rereplication induced by these manipulations is however uneven and incomplete, suggesting that one or more mechanisms restrain rereplication once it begins. We find that both Cdt1 and Cdc6 are degraded in geminin-depleted cells. We further show that Cdt1 degradation in cells that have rereplicated requires the PCNA binding site of Cdt1 and the Cul4 DDB1 ubiquitin ligase, and Cdt1 can induce its own degradation when overproduced. Cdc6 degradation in geminin-depleted cells requires Huwe1, the ubiquitin ligase that regulates Cdc6 after DNA damage. Moreover, perturbations that specifically disrupt Cdt1 and Cdc6 degradation in response to DNA damage exacerbate rereplication when combined with geminin depletion, and this enhanced rereplication occurs in both human cells and in Drosophila melanogaster cells. We conclude that rereplication-associated DNA damage triggers Cdt1 and Cdc6 ubiquitination and destruction, and propose that this pathway represents an evolutionarily conserved mechanism that minimizes the extent of rereplication.One of the critical events in the cell division cycle is complete and precise duplication of the genome. In eukaryotic cells, origins of DNA replication acquire replication competence through the assembly of a prereplication complex (preRC) 3 in the G 1 phase of the cell cycle. PreRCs are assembled by the sequential origin binding of the origin recognition complex (ORC), Cdc6, Cdt1, and the minichromosome maintenance complex (MCM). Origins harboring preRCs are licensed for replication but do not initiate DNA synthesis until S phase begins and the Cdc7 and Cdk2 kinases are activated (1-3). The large genomes of metazoan cells necessitate the utilization of thousands of origins, but each origin that initiates DNA synthesis must do so only once. Failure to maintain this control has been linked to genome instability and oncogenesis (4, 5), but the cellular consequences of rereplication are not fully understood. Multiple regulatory mechanisms operate to ensure that any origins that have "fired" do not fire again by blocking preRC assembly after the G 1 to S phase transition. Among the most important of these mechanisms are degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Geminin depletion, overexpression of Cdt1, or overexpression of Cdc6 causes rereplication, which ultimately triggers a DNA damage response (6 -11) and can promote tumorigenesis (4, 5). Rereplication induced by these manipulations is however incomplete, with the extent of rereplication varying widely among different cell lines. These observations suggest that in addition to the mechanisms that bloc...

A major challenge each human cell-division cycle is to ensure that DNA replication origins do not initiate more than once, a phenomenon known as re-replication. Acute deregulation of replication control ultimately causes extensive DNA damage, cell-cycle checkpoint activation and cell death whereas moderate deregulation promotes genome instability and tumorigenesis. In the absence of detectable increases in cellular DNA content however, it has been difficult to directly demonstrate re-replication or to determine if the ability to re-replicate is restricted to a particular cell-cycle phase. Using an adaptation of DNA fiber spreading we report the direct detection of re-replication on single DNA molecules from human chromosomes. Using this method we demonstrate substantial re-replication within 1 h of S phase entry in cells overproducing the replication factor, Cdt1. Moreover, a comparison of the HeLa cancer cell line to untransformed fibroblasts suggests that HeLa cells produce replication signals consistent with low-level re-replication in otherwise unperturbed cell cycles. Re-replication after depletion of the Cdt1 inhibitor, geminin, in an untransformed fibroblast cell line is undetectable by standard assays but readily quantifiable by DNA fiber spreading analysis. Direct evaluation of re-replicated DNA molecules will promote increased understanding of events that promote or perturb genome stability.