C. elegans CUL-4 prevents rereplication by promoting the nuclear export of CDC-6 via a CKI-1-dependent pathway

Genome stability requires that genomic DNA is replicated only once per cell cycle. The replication-licensing system ensures that the formation of prereplicative complexes is temporally separated from the initiation of DNA replication [1-4]. The replication-licensing factors Cdc6 and Cdt1 are required for the assembly of prereplicative complexes during G1 phase. During S phase, metazoan Cdt1 is targeted for degradation by the CUL4 ubiquitin ligase [5-8], and vertebrate Cdc6 is translocated from the nucleus to the cytoplasm [9, 10]. However, because residual vertebrate Cdc6 remains in the nucleus throughout S phase [10-13], it has been unclear whether Cdc6 translocation to the cytoplasm prevents rereplication [1, 2, 14]. The inactivation of C. elegans CUL-4 is associated with dramatic levels of DNA rereplication [5]. Here, we show that C. elegans CDC-6 is exported from the nucleus during S phase in response to the phosphorylation of multiple CDK sites. CUL-4 promotes the phosphorylation and subsequent translocation of CDC-6 via negative regulation of the CDK-inhibitor CKI-1. Rereplication can be induced by coexpression of nonexportable CDC-6 with nondegradable CDT-1, indicating that redundant regulation of CDC-6 and CDT-1 prevents rereplication. This demonstrates that CDC-6 translocation is critical for preventing rereplication and that CUL-4 independently controls both replication-licensing factors.     

PCNA is a cofactor for Cdt1 degradation by CUL4/DDB1-mediated N-terminal ubiquitination

Cdt1, a protein essential in G1 for licensing of origins for DNA replication, is inhibited in S-phase, both by binding to geminin and degradation by proteasomes. Cdt1 is also degraded after DNA damage to stop licensing of new origins until after DNA repair. Phosphorylation of Cdt1 by cyclin-dependent kinases promotes its binding to SCF-Skp2 E3 ubiquitin ligase, but the Cdk2/Skp2-mediated pathway is not essential for the degradation of Cdt1. Here we show that the N terminus of Cdt1 contains a second degradation signal that is active after DNA damage and in S-phase and is dependent on the interaction of Cdt1 with proliferating cell nuclear antigen (PCNA) through a PCNA binding motif. The degradation involves N-terminal ubiquitination and requires Cul4 and Ddb1 proteins, components of an E3 ubiquitin ligase implicated in protein degradation after DNA damage. Therefore PCNA, the matchmaker for many proteins involved in DNA and chromatin metabolism, also serves to promote the targeted degradation of associated proteins in S-phase or after DNA damage.     

DDB1 maintains genome integrity through regulation of Cdt1

DDB1, a component of a Cul4A ubiquitin ligase complex, promotes nucleotide excision repair (NER) and regulates DNA replication. We have investigated the role of human DDB1 in maintaining genome stability. DDB1-depleted cells accumulate DNA double-strand breaks in widely dispersed regions throughout the genome and have activated ATM and ATR cell cycle checkpoints. Depletion of Cul4A yields similar phenotypes, indicating that an E3 ligase function of DDB1 is important for genome maintenance. In contrast, depletion of DDB2, XPA, or XPC does not cause activation of DNA damage checkpoints, indicating that defects in NER are not involved. One substrate of DDB1-Cul4A that is crucial for preventing genome instability is Cdt1. DDB1-depleted cells exhibit increased levels of Cdt1 protein and rereplication, despite containing other Cdt1 regulatory mechanisms. The rereplication, accumulation of DNA damage, and activation of checkpoint responses in DDB1-depleted cells require entry into S phase and are partially, but not completely, suppressed by codepletion of Cdt1. Therefore, DDB1 prevents DNA lesions from accumulating in replicating human cells, in part by regulating Cdt1 degradation.     

The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCF^Cdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.     

Two different replication factor C proteins, Ctf18 and RFC1, separately control PCNA-CRL4Cdt2-mediated Cdt1 proteolysis during S phase and following UV irradiation

Recent work identified the E3 ubiquitin ligase CRL4(Cdt2) as mediating the timely degradation of Cdt1 during DNA replication and following DNA damage. In both cases, proliferating cell nuclear antigen (PCNA) loaded on chromatin mediates the CRL4(Cdt2)-dependent proteolysis of Cdt1. Here, we demonstrate that while replication factor C subunit 1 (RFC1)-RFC is required for Cdt1 degradation after UV irradiation during the nucleotide excision repair process, another RFC complex, Ctf18-RFC, which is known to be involved in the establishment of cohesion, has a key role in Cdt1 degradation in S phase. Cdt1 segments having only the degron, a specific sequence element in target protein for ubiquitination, for CRL4(Cdt2) were stabilized during S phase in Ctf18-depleted cells. Additionally, endogenous Cdt1 was stabilized when both Skp2 and Ctf18 were depleted. Since a substantial amount of PCNA was detected on chromatin in Ctf18-depleted cells, Ctf18 is required in addition to loaded PCNA for Cdt1 degradation in S phase. Our data suggest that Ctf18 is involved in recruiting CRL4(Cdt2) to PCNA foci during S phase. Ctf18-mediated Cdt1 proteolysis occurs independent of cohesion establishment, and depletion of Ctf18 potentiates rereplication. Our findings indicate that individual RFC complexes differentially control CRL4(Cdt2)-dependent proteolysis of Cdt1 during DNA replication and repair.     

Depletion of licensing inhibitor geminin causes centrosome overduplication and mitotic defects

Metazoans limit origin firing to once per cell cycle by oscillations in cyclin-dependent kinases and the replication licensing inhibitor geminin. Geminin inhibits pre-replication complex assembly by preventing Cdt1 from recruiting the minichromosome maintenance proteins to chromatin. Geminin depletion results in genomic over-replication in Drosophila and human cell lines. Here, we show that loss of geminin affects other cell cycle-dependent events in addition to DNA replication. Geminin inactivation causes centrosome overduplication without passage through mitosis in human normal and cancer cells. Centrosomes are microtubule-organizing centres that are duplicated during S phase and have an important role in the fidelity of chromosome transmission by nucleating the mitotic spindle. Consistent with this, geminin-depleted cells show multiple mitotic defects, including multipolar spindles, when driven into mitosis by checkpoint abrogation. These results show that the consequences of geminin loss exceed its immediate role in DNA replication and extend to promoting chromosome mis-segregation in mitosis.     

The regulated association of Cdt1 with minichromosome maintenance proteins and Cdc6 in mammalian cells

Chromosomal DNA replication requires the recruitment of the six-subunit minichromosome maintenance (Mcm) complex to chromatin through the action of Cdc6 and Cdt1. Although considerable work has described the functions of Cdc6 and Cdt1 in yeast and biochemical systems, evidence that their mammalian counterparts are subject to distinct regulation suggests the need to further explore the molecular relationships involving Cdc6 and Cdt1. Here we demonstrate that Cdc6 and Cdt1 are mutually dependent on one another for loading Mcm complexes onto chromatin in mammalian cells. The association of Cdt1 with Mcm2 is regulated by cell growth. Mcm2 prepared from quiescent cells associates very weakly with Cdt1, whereas Mcm2 from serum-stimulated cells associates with Cdt1 much more efficiently. Cdc6, which normally accumulates as cells progress from quiescence into G(1), is capable of inducing the binding of Mcm2 to Cdt1 when ectopically expressed in quiescent cells. We further show that Cdc6 physically associates with Cdt1 via its N-terminal noncatalytic domain, a region we had previously shown to be essential for Cdc6 function. Cdt1 activity is inhibited by the geminin protein, and we provide evidence that the mechanism of this inhibition involves blocking the binding of Cdt1 to both Mcm2 and Cdc6. These results identify novel molecular functions for both Cdc6 and geminin in controlling the association of Cdt1 with other components of the replication apparatus and indicate that the association of Cdt1 with the Mcm complex is controlled as cells exit and reenter the cell cycle.     

A Cdt1-geminin complex licenses chromatin for DNA replication and prevents rereplication during S phase in Xenopus

Initiation of DNA synthesis involves the loading of the MCM2-7 helicase onto chromatin by Cdt1 (origin licensing). Geminin is thought to prevent relicensing by binding and inhibiting Cdt1. Here we show, using Xenopus egg extracts, that geminin binding to Cdt1 is not sufficient to block its activity and that a Cdt1-geminin complex licenses chromatin, but prevents rereplication, working as a molecular switch at replication origins. We demonstrate that geminin is recruited to chromatin already during licensing, while bulk geminin is recruited at the onset of S phase. A recombinant Cdt1-geminin complex binds chromatin, interacts with the MCM2-7 complex and licenses chromatin once per cell cycle. Accordingly, while recombinant Cdt1 induces rereplication in G1 or G2 and activates an ATM/ATR-dependent checkpoint, the Cdt1-geminin complex does not. We further demonstrate that the stoichiometry of the Cdt1-geminin complex regulates its activity. Our results suggest a model in which the MCM2-7 helicase is loaded onto chromatin by a Cdt1-geminin complex, which is inactivated upon origin firing by binding additional geminin. This origin inactivation reaction does not occur if only free Cdt1 is present on chromatin.     

Identification of novel human Cdt1-binding proteins by a proteomics approach: proteolytic regulation by APC/CCdh1

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIalpha, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/C(Cdh1), SNF2H, topoisomerase I and IIalpha, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/C(Cdh1) indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCF(Skp2) and cullin4-based ubiquitin ligases, APC/C(Cdh1) is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.     

An evolutionarily conserved function of proliferating cell nuclear antigen for Cdt1 degradation by the Cul4-Ddb1 ubiquitin ligase in response to DNA damage

The DNA replication licensing factor Cdt1 is degraded by the ubiquitin-proteasome pathway during S phase of the cell cycle, to ensure one round of DNA replication during each cell division and in response to DNA damage to halt DNA replication. Constitutive expression of Cdt1 causes DNA re-replication and is associated with the development of a subset of human non-small cell-lung carcinomas. In mammalian cells, DNA damage-induced Cdt1 degradation is catalyzed by the Cul4-Ddb1-Roc1 E3 ubiquitin ligase. We report here that overexpression of the proliferating cell nuclear antigen (PCNA) inhibitory domain from the CDK inhibitors p21 and p57, but not the CDK-cyclin inhibitory domain, blocked Cdt1 degradation in cultured mammalian cells after UV irradiation. In vivo soluble Cdt1 and PCNA co-elute by gel filtration and associate with each other physically. Silencing PCNA in cultured mammalian cells or repression of pcn1 expression in fission yeast blocked Cdt1 degradation in response to DNA damage. Unexpectedly, deletion of Ddb1 in fission yeast cells also accumulated Cdt1 in the absence of DNA damage. We suggest that the Cul4-Ddb1 ligase evolved to ubiquitinate Cdt1 during normal cell growth as well as in response to DNA damage and a separate E3 ligase, possibly SCF(Skp2), evolved to either share or take over the function of Cdt1 ubiquitination during normal cell growth and that PCNA is involved in mediating Cdt1 degradation by the Cul4-Ddb1 ligase in response to DNA damage.     

The Caenorhabditis elegans replication licensing factor CDT-1 is targeted for degradation by the CUL-4/DDB-1 complex

The replication of genomic DNA is strictly regulated to occur only once per cell cycle. This regulation centers on the temporal restriction of replication licensing factor activity. Two distinct ubiquitin ligase (E3) complexes, CUL4/DDB1 and SCF(Skp2), have been reported to target the replication licensing factor Cdt1 for ubiquitin-mediated proteolysis. However, it is unclear to what extent these two distinct Cdt1 degradation pathways are conserved. Here, we show that Caenorhabditis elegans DDB-1 is required for the degradation of CDT-1 during S phase. DDB-1 interacts specifically with CUL-4 but not with other C. elegans cullins. A ddb-1 null mutant exhibits extensive DNA rereplication in postembryonic BLAST cells, similar to what is observed in cul-4(RNAi) larvae. DDB-1 physically associates with CDT-1, suggesting that CDT-1 is a direct substrate of the CUL-4/DDB-1 E3 complex. In contrast, a deletion mutant of the C. elegans Skp2 ortholog, skpt-1, appears overtly wild type with the exception of an impenetrant gonad migration defect. There is no appreciable role for SKPT-1 in the degradation of CDT-1 during S phase, even in a sensitized ddb-1 mutant background. We propose that the CUL-4/DDB-1 ubiquitin ligase is the principal E3 for regulating the extent of DNA replication in C. elegans.     

Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage

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.     

Regulation of germline stem cell proliferation downstream of nutrient sensing

In eukaryotic cells, replication of genomic DNA initiates from multiple replication origins distributed on multiple chromosomes. To ensure that each origin is activated precisely only once during each S phase, a system has evolved which features periodic assembly and disassembly of essential pre-replication complexes (pre-RCs) at replication origins. The pre-RC assembly reaction involves the loading of a presumptive replicative helicase, the MCM2-7 complexes, onto chromatin by the origin recognition complex (ORC) and two essential factors, CDC6 and Cdt1. The eukaryotic cell cycle is driven by the periodic activation and inactivation of cyclin-dependent kinases (Cdks) and assembly of pre-RCs can only occur during the low Cdk activity period from late mitosis through G1 phase, with inappropriate re-assembly suppressed during S, G2, and M phases. It was originally suggested that inhibition of Cdt1 function after S phase in vertebrate cells is due to geminin binding and that Cdt1 hyperfunction resulting from Cdt1-geminin imbalance induces re-replication. However, recent progress has revealed that Cdt1 activity is more strictly regulated by two other mechanisms in addition to geminin: (1) functional and SCF^Skp2-mediated proteolytic regulation through phosphorylation by Cdks; and (2) replication-coupled proteolysis mediated by the Cullin4-DDB1^Cdt2 ubiquitin ligase and PCNA, an eukaryotic sliding clamp stimulating replicative DNA polymerases. The tight regulation implies that Cdt1 control is especially critical for the regulation of DNA replication in mammalian cells. Indeed, Cdt1 overexpression evokes chromosomal damage even without re-replication. Furthermore, deregulated Cdt1 induces chromosomal instability in normal human cells. Since Cdt1 is overexpressed in cancer cells, this could be a new molecular mechanism leading to carcinogenesis. In this review, recent insights into Cdt1 function and regulation in mammalian cells are discussed.     

A p53-dependent checkpoint pathway prevents rereplication

Eukaryotic cells control the initiation of DNA replication so that origins that have fired once in S phase do not fire a second time within the same cell cycle. Failure to exert this control leads to genetic instability. Here we investigate how rereplication is prevented in normal mammalian cells and how these mechanisms might be overcome during tumor progression. Overexpression of the replication initiation factors Cdt1 and Cdc6 along with cyclin A-cdk2 promotes rereplication in human cancer cells with inactive p53 but not in cells with functional p53. A subset of origins distributed throughout the genome refire within 2-4 hr of the first cycle of replication. Induction of rereplication activates p53 through the ATM/ATR/Chk2 DNA damage checkpoint pathways. p53 inhibits rereplication through the induction of the cdk2 inhibitor p21. Therefore, a p53-dependent checkpoint pathway is activated to suppress rereplication and promote genetic stability.     

Cell cycle regulation of the licensing activity of Cdt1 in Xenopus laevis

Cdt1 is a conserved replication factor required in licensing the chromosome for a single round of DNA synthesis. The activity of Cdt1 is inhibited by geminin. The mechanism by which geminin interferes with Cdt1 activity is unknown. It is thought that geminin binds to and sequestrate Cdt1. We show that geminin does not interfere with the chromatin association of Cdt1 and that inhibition of DNA synthesis by geminin is observed following its accumulation on chromatin. The binding of geminin to chromatin has been investigated during S phase. We demonstrate that loading of geminin onto chromatin requires Cdt1, suggesting that geminin is targeted at replication origins. We also show that geminin binds chromatin at the transition from the pre-replication to pre-initiation complexes, which overlaps with the release of Cdt1. This regulation is strikingly different from that observed in somatic cells where the chromatin binding of these proteins is mutually exclusive. In contrast to somatic cells, we further show that geminin is stable during the early embryonic cell cycles. These results suggest a specific regulation of origin firing adapted to the rapid cell cycles of Xenopus and indicate that periodic degradation of geminin is not relevant to licensing during embryonic development.     

Geminin deletion in mouse oocytes results in impaired embryo development and reduced fertility

Geminin controls proper centrosome duplication, cell division, and differentiation. We investigated the function of geminin in oogenesis, fertilization, and early embryo development by deleting the geminin gene in oocytes from the primordial follicle stage. Oocyte-specific disruption of geminin results in low fertility in mice. Even though there was no evident anomaly of oogenesis, oocyte meiotic maturation, natural ovulation, or fertilization, early embryo development and implantation were impaired. The fertilized eggs derived from mutant mice showed developmental delay, and many were blocked at the late zygote stage. Cdt1 protein was decreased, whereas Chk1 and H2AX phosphorylation was increased, in fertilized eggs after geminin depletion. Our results suggest that disruption of maternal geminin may decrease Cdt1 expression and cause DNA rereplication, which then activates the cell cycle checkpoint and DNA damage repair and thus impairs early embryo development.     

Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding

The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.