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Geminin has been proposed to coordinate cell cycle and differentiation events through balanced interactions with the cell cycle regulator Cdt1 and with homeobox transcription factors and chromatin remodeling activities implicated in cell fate decisions. Here we show that Geminin is cleaved in primary cells and cancer cell lines induced to undergo apoptosis by a variety of stimuli. Geminin targeting is mediated by caspase-3 both in vivo and in vitro. Two sites at the carboxyl terminus of Geminin (named C1 and C2) are cleaved by the caspase, producing truncated forms of Geminin. We provide evidence that Geminin cleavage is regulated by phosphorylation. Casein kinase II alters Geminin cleavage at site C1 in vitro, whereas mutating phosphorylation competent Ser/Thr residues proximal to site C1 affects Geminin cleavage in vivo. We show that truncated Geminin produced by cleavage at C1 can promote apoptosis. In contrast, Geminin cleaved at site C2 has lost the ability to interact with Brahma (Brm), a catalytic subunit of the SWI/SNF chromatin remodeling complex, while binding efficiently to Cdt1, indicating that targeting of Geminin during apoptosis differentially affects interactions with its binding partners.

To maintain genome integrity, eukaryotic cells initiate DNA replication once per cell cycle after assembling prereplicative complexes (preRCs) on chromatin at the end of mitosis and during G1. In S phase, preRCs are disassembled, precluding initiation of another round of replication. Cdt1 is a key member of the preRC and its correct regulation via proteolysis and by its inhibitor Geminin is essential to prevent premature re-replication. Using quantitative fluorescence microscopy, we study the interactions of Cdt1 with chromatin and Geminin in living cells. We find that Cdt1 exhibits dynamic interactions with chromatin throughout G1 phase and that the protein domains responsible for chromatin and Geminin interactions are separable. Contrary to existing in vitro data, we show that Cdt1 simultaneously binds Geminin and chromatin in vivo, thereby recruiting Geminin onto chromatin. We propose that dynamic Cdt1-chromatin associations and the recruitment of Geminin to chromatin provide spatio-temporal control of the licensing process.

DNA replication, the process of duplication of a cell's genetic content, must be carried out with great precision every time the cell divides, so that genetic information is preserved. Control mechanisms must ensure that every base of the genome is replicated within the allocated time (S-phase) and only once per cell cycle, thereby safeguarding genomic integrity. In eukaryotes, replication starts from many points along the chromosome, termed origins of replication, and then proceeds continuously bidirectionally until an opposing moving fork is encountered. In contrast to bacteria, where a specific site on the genome serves as an origin in every cell division, in most eukaryotes origin selection appears highly stochastic: many potential origins exist, of which only a subset is selected to fire in any given cell, giving rise to an apparently random distribution of initiation events across the genome. Origin states change throughout the cell cycle, through the ordered formation and modification of origin-associated multisubunit protein complexes. State transitions are governed by fluctuations of cyclin-dependent kinase (CDK) activity and guards in these transitions ensure system memory. We present here DNA replication dynamics, emphasizing recent data from the fission yeast Schizosaccharomyces pombe, and discuss how robustness may be ensured in spite of (or even assisted by) system randomness., (Copyright 2006 John Wiley & Sons, Ltd.)

Replication licensing is carefully regulated to restrict replication to once in a cell cycle. In higher eukaryotes, regulation of the licensing factor Cdt1 by proteolysis and Geminin is essential to prevent re-replication. We show here that the N-terminal 100 amino acids of human Cdt1 are recognized for proteolysis by two distinct E3 ubiquitin ligases during S-G2 phases. Six highly conserved amino acids within the 10 first amino acids of Cdt1 are essential for DDB1-Cul4-mediated proteolysis. This region is also involved in proteolysis following DNA damage. The second E3 is SCF-Skp2, which recognizes the Cy-motif-mediated Cyclin E/A-cyclin-dependent kinase-phosphorylated region. Consistently, in HeLa cells cosilenced of Skp2 and Cul4, Cdt1 remained stable in S-G2 phases. The Cul4-containing E3 is active during ongoing replication, while SCF-Skp2 operates both in S and G2 phases. PCNA binds to Cdt1 through the six conserved N-terminal amino acids. PCNA is essential for Cul4- but not Skp2-directed degradation during DNA replication and following ultraviolet-irradiation. Our data unravel multiple distinct pathways regulating Cdt1 to block re-replication.

Replication licensing ensures once per cell cycle replication and is essential for genome stability. Overexpression of two key licensing factors, Cdc6 and Cdt1, leads to overreplication and chromosomal instability (CIN) in lower eukaryotes and recently in human cell lines. In this report, we analyzed hCdt1, hCdc6, and hGeminin, the hCdt1 inhibitor expression, in a series of non-small-cell lung carcinomas, and investigated for putative relations with G(1)/S phase regulators, tumor kinetics, and ploidy. This is the first study of these fundamental licensing elements in primary human lung carcinomas. We herein demonstrate elevated levels (more than fourfold) of hCdt1 and hCdc6 in 43% and 50% of neoplasms, respectively, whereas aberrant expression of hGeminin was observed in 49% of cases (underexpression, 12%; overexpression, 37%). hCdt1 expression positively correlated with hCdc6 and E2F-1 levels (P = 0.001 and P = 0.048, respectively). Supportive of the observed link between E2F-1 and hCdt1, we provide evidence that E2F-1 up-regulates the hCdt1 promoter in cultured mammalian cells. Interestingly, hGeminin overexpression was statistically related to increased hCdt1 levels (P = 0.025). Regarding the kinetic and ploidy status of hCdt1- and/or hCdc6-overexpressing tumors, p53-mutant cases exhibited significantly increased tumor growth values (Growth Index; GI) and aneuploidy/CIN compared to those bearing intact p53 (P = 0.008 for GI, P = 0.001 for CIN). The significance of these results was underscored by the fact that the latter parameters were independent of p53 within the hCdt1-hCdc6 normally expressing cases. Cumulatively, the above suggest a synergistic effect between hCdt1-hCdc6 overexpression and mutant-p53 over tumor growth and CIN in non-small-cell lung carcinomas.

The DNA replication licensing system ensures that chromosomal DNA is replicated precisely once before cell division occurs. A DNA helicase must be loaded on origin DNA for replication to initiate. Considerable evidence suggests that the MCM complex acts as a replicative helicase in eukaryotes. When the MCM complex is loaded on the chromatin, the replication origin is formally defined as being licensed for replication. Licensing takes place several hours before origins are activated to undergo replication in S-phase. Genetic and biochemical studies show that the licensing process is well conserved in eukaryotes. Cyclin Dependent Kinases (CDKs), the master regulators of the cell cycle, coordinate the initiation of the two key cell cycle events, replication of DNA and its segregation at mitosis. Eukaryotes have developed complex regulatory mechanisms to ensure that origin licensing is coordinated with these events so that genome integrity is preserved during successive cell divisions.

Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3-6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells.

Licensing of replication origins is carefully regulated in a cell cycle to maintain genome integrity. Using an in vivo ubiquitination assay and temperature-sensitive cell lines we demonstrate that Cdt1 in mammalian cells is degraded through ubiquitin-dependent proteolysis in S-phase. siRNA experiments for Geminin indicate that Cdt1 is degraded in the absence of Geminin. The N terminus of Cdt1 is required for its nuclear localization, associates with cyclin A, but is dispensable for the association of Cdt1 with Geminin in cells. This region is responsible for proteolysis of Cdt1 in S-phase. On the other hand, the N terminus-truncated Cdt1 is stable in S-phase, and associates with the licensing inhibitor, Geminin. High level expression of this form of Cdt1 brings about cells having higher DNA content. Proteasome inhibitors stabilize Cdt1 in S-phase, and the protein is detected in the nucleus in a complex with Geminin. This form of Cdt1 associates with chromatin as tightly as that of G1-cells, when no Geminin is detected. Our data show that proteolysis and Geminin binding independently inactivate Cdt1 after the onset of S-phase to prevent re-replication.

To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Its control has been uncovered by the discovery of the CDKs (cyclin dependent kinases) as master regulators of the cell cycle and the initiator proteins of DNA replication, such as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At the end of mitosis, the MCM complex is loaded on to chromatin with the aid of ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs, together with the Cdc7 kinase, trigger the initiation of replication, recruiting the DNA replicating enzymes on sites of replication. The activated MCM complex appears to play a key role in the DNA unwinding step, acting as a replicating helicase and moves along with the replication fork, at the same time bringing the origins to the unlicensed state. The cycling of CDK activity in the cell cycle separates the two states of replication origins, the licensed state in G1-phase and the unlicensed state for the rest of the cell cycle. Only when CDK drops at the completion of mitosis, is the restriction on licensing relieved and a new round of replication is allowed. Such a CDK-regulated licensing control is conserved from yeast to higher eukaryotes, and ensures that DNA replication takes place only once in a cycle. Xenopus laevis and mammalian cells have an additional system to control licensing. Geminin, whose degradation at the end of mitosis is essential for a new round of licensing, has been shown to bind Cdt1 and negatively regulate it, providing a new insight into the regulation of DNA replication in higher eukaryotes.

Cdc18/Cdc6 and Cdt1 are essential initiation factors for DNA replication. In this paper we show that expression of Cdc18 in fission yeast G2 cells is sufficient to override the controls that ensure one S phase per cell cycle. Cdc18 expression in G2 induces DNA synthesis by re-firing replication origins and recruiting the MCM Cdc21 to chromatin in the presence of low levels of Cdt1. However, when Cdt1 is expressed together with Cdc18 in G2, cells undergo very rapid, uncontrolled DNA synthesis, accumulating DNA contents of 64C or more. Our data suggest that Cdt1 may potentiate re-replication by inducing origins to fire more persistently, possibly by stabilizing Cdc18 on chromatin. In addition, low level expression of a mutant form of Cdc18 that cannot be phosphorylated by cyclin-dependent kinases is not sufficient to induce replication in G2, but does so only when co-expressed with Cdt1. Thus, regulation of both Cdc18 and Cdt1 in G2 plays a crucial role in preventing the re-initiation of DNA synthesis until the next cell cycle.

A cell ensures that its genome is replicated only once during its division cycle through a process called licensing. In an enlightening Perspective, Lygerou and Nurse explain how binding of the Geminin protein to Cdt1 blocks the binding of licensing factors to chromatin, inhibiting the onset of S phase (Wohlschlegel et al.).

To maintain genome stability in eukaryotic cells, DNA is licensed for replication only after the cell has completed mitosis, ensuring that DNA synthesis (S phase) occurs once every cell cycle. This licensing control is thought to require the protein Cdc6 (Cdc18 in fission yeast) as a mediator for association of minichromosome maintenance (MCM) proteins with chromatin. The control is overridden in fission yeast by overexpressing Cdc18 (ref. 11) which leads to continued DNA synthesis in the absence of mitosis. Other factors acting in this control have been postulated and we have used a re-replication assay to identify Cdt1 (ref. 14) as one such factor. Cdt1 cooperates with Cdc18 to promote DNA replication, interacts with Cdc18, is located in the nucleus, and its concentration peaks as cells finish mitosis and proceed to S phase. Both Cdc18 and Cdt1 are required to load the MCM protein Cdc21 onto chromatin at the end of mitosis and this is necessary to initiate DNA replication. Genes related to Cdt1 have been found in Metazoa and plants (A. Whitaker, I. Roysman and T. Orr-Weaver, personal communication), suggesting that the cooperation of Cdc6/Cdc18 with Cdt1 to load MCM proteins onto chromatin may be a generally conserved feature of DNA licensing in eukaryotes.

The assembly pathway of spliceosomal snRNPs in yeast is poorly understood. We devised a screen to identify mutations blocking the assembly of newly synthesized U4 snRNA into a functional snRNP. Fifteen mutant strains failing either to accumulate the newly synthesized U4 snRNA or to assemble a U4/U6 particle were identified and categorized into 13 complementation groups. Thirteen previously identified splicing-defective prp mutants were also assayed for U4 snRNP assembly defects. Mutations in the U4/U6 snRNP components Prp3p, Prp4p, and Prp24p led to disassembly of the U4/U6 snRNP particle and degradation of the U6 snRNA, while prp17-1 and prp19-1 strains accumulated free U4 and U6 snRNA. A detailed analysis of a newly identified mutant, the sad1-1 mutant, is presented. In addition to having the snRNP assembly defect, the sad1-1 mutant is severely impaired in splicing at the restrictive temperature: the RP29 pre-mRNA strongly accumulates and splicing-dependent production of beta-galactosidase from reporter constructs is abolished, while extracts prepared from sad1-1 strains fail to splice pre-mRNA substrates in vitro. The sad1-1 mutant is the only splicing-defective mutant analyzed whose mutation preferentially affects assembly of newly synthesized U4 snRNA into the U4/U6 particle. SAD1 encodes a novel protein of 52 kDa which is essential for cell viability. Sad1p localizes to the nucleus and is not stably associated with any of the U snRNAs. Sad1p contains a putative zinc finger and is phylogenetically highly conserved, with homologues identified in human, Caenorhabditis elegans, Arabidospis, and Drosophila.

Very few of the enzymes required for eukaryotic precursor ribosomal RNA (pre-rRNA) processing have been identified. Ribonuclease (RNase) MRP was characterized as a nuclease that cleaves mitochondrial replication primers, but it is predominantly nucleolar. Previous genetic evidence revealed that this ribonucleoprotein is required, directly or indirectly, for cleavage of the yeast pre-rRNA in vivo at site A3. Here, an in vitro processing system that accurately reproduces this cleavage is described. Biochemical purification and the use of extracts depleted of the MRP RNA demonstrate that endonucleolytic cleavage of the pre-rRNA is directly mediated by RNase MRP. This establishes a role for RNase MRP in the nucleolus.

While screening for genes that affect the synthesis of yeast snRNPs, we identified a thermosensitive mutant that abolishes the production of a reporter snRNA at the non-permissive temperature. This mutant defines a new gene, named BDF1. In a bdf1-1 strain, the reporter snRNA synthesized before the temperature shift remains stable at the non-permissive temperature. This demonstrates that the BDF1 gene affects the synthesis rather than the stability of the reporter snRNA and suggests that the BDF1 gene encodes a transcription factor. BDF1 is present in single copy on yeast chromosome XII, and is important for normal vegetative growth but not essential for cell viability. bdf1 null mutants share common phenotypes with several mutants affecting general transcription and are defective in snRNA production. BDF1 encodes a protein of 687 amino-acids containing two copies of the bromodomain, a motif also present in other transcription factors as well as a new conserved domain, the ET domain, also present in Drosophila and human proteins.