L2DTL/CDT2 and PCNA Interact with p53 and Regulate p53 Polyubiquitination and Protein Stability through MDM2 and CUL4A/DDB1 Complexes

The CUL4-ROC1 E3 ligase complex regulates genome stability, replication, and cell cycle progression. A novel WD40 domain-containing protein, L2DTL, and PCNA were identified as proteins associated with CUL4/DDB1 complexes. Inactivation of CUL4A, L2DTL, PCNA, DDB1, or ROC1 induced p53 stabilization and growth arrest. L2DTL, PCNA, and DDB1/CUL4A complexes were found to physically interact with p53 tumor suppressor and its regulator MDM2/HDM2. The isolated CUL4A complexes display potent and robust polyubiquitination activity towards p53 and this activity is dependent on L2DTL, PCNA, DDB1, ROC1, and MDM2/HDM2. We also found that the interaction between p53 and CUL4 complex is regulated by DNA damage. Our data further showed that MDM2/HDM2 is rapidly proteolyzed in response to UV irradiation and this process is regulated by CUL4/DDB1 and PCNA. Our studies demonstrate that PCNA, L2DTL, and the DDB1-CUL4A complex play critical and differential roles in regulating the protein stability of p53 and MDM2/HDM2 in unstressed and stressed cells.     

CUL4-DDB1 ubiquitin ligase interacts with multiple WD40-repeat proteins and regulates histone methylation

The CUL4-DDB1-ROC1 ubiquitin E3 ligase regulates cell-cycle progression, replication and DNA damage response. However, the substrate-specific adaptors of this ligase remain uncharacterized. Here, we show that CUL4-DDB1 complexes interact with multiple WD40-repeat proteins (WDRs) including TLE1-3, WDR5, L2DTL (also known as CDT2) and the Polycomb-group protein EED (also known as ESC). WDR5 and EED are core components of histone methylation complexes that are essential for histone H3 methylation and epigenetic control at K4 or K9 and K27, respectively, whereas L2DTL regulates CDT1 proteolysis after DNA damage through CUL4-DDB1 (ref. 8). We found that CUL4A-DDB1 interacts with H3 methylated mononucleosomes and peptides. Inactivation of either CUL4 or DDB1 impairs these histone modifications. However, loss of WDR5 specifically affects histone H3 methylation at K4 but not CDT1 degradation, whereas inactivation of L2DTL prevents CDT1 degradation but not histone methylation. Our studies suggest that CUL4-DDB1 ligases use WDR proteins as molecular adaptors for substrate recognition, and modulate multiple biological processes through ubiquitin-dependent proteolysis.     

Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage

Cullins assemble a potentially large number of ubiquitin ligases by binding to the RING protein ROC1 to catalyse polyubiquitination, as well as binding to various specificity factors to recruit substrates. The Cul4A gene is amplified in human breast and liver cancers, and loss-of-function of Cul4 results in the accumulation of the replication licensing factor CDT1 in Caenorhabditis elegans embryos and ultraviolet (UV)-irradiated human cells. Here, we report that human UV-damaged DNA-binding protein DDB1 associates stoichiometrically with CUL4A in vivo, and binds to an amino-terminal region in CUL4A in a manner analogous to SKP1, SOCS and BTB binding to CUL1, CUL2 and CUL3, respectively. As with SKP1-CUL1, the DDB1-CUL4A association is negatively regulated by the cullin-associated and neddylation-dissociated protein, CAND1. Recombinant DDB1 and CDT1 bind directly to each other in vitro, and ectopically expressed DDB1 bridges CDT1 to CUL4A in vivo. Silencing DDB1 prevented UV-induced rapid CDT1 degradation in vivo and CUL4A-mediated CDT1 ubiquitination in vitro. We suggest that DDB1 targets CDT1 for ubiquitination by a CUL4A-dependent ubiquitin ligase, CDL4A(DDB1), in response to UV irradiation.     

Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint

Genomic integrity is maintained by checkpoints that guard against undesired replication after DNA damage. Here, we show that CDT1, a licensing factor of the pre-replication complex (preRC), is rapidly proteolysed after UV- or gamma-irradiation. The preRC assembles on replication origins at the end of mitosis and during G1 to license DNA for replication in S phase. Once the origin recognition complex (ORC) binds to origins, CDC6 and CDT1 associate with ORC and promote loading of the MCM2-7 proteins onto chromatin, generating the preRC. We show that radiation-mediated CDT1 proteolysis is independent of ATM and CHK2 and can occur in G1-phase cells. Loss of the COP9-signalosome (CSN) or CUL4-ROC1 complexes completely suppresses CDT1 proteolysis. CDT1 is specifically polyubiquitinated by CUL4 complexes and the interaction between CDT1 and CUL4 is regulated in part by gamma-irradiation. Our study reveals an evolutionarily conserved and uncharacterized G1 checkpoint that induces CDT1 proteolysis by the CUL4-ROC1 ubiquitin E3 ligase and CSN complexes in response to DNA damage.     

Cellular concentrations of DDB2 regulate dynamic binding of DDB1 at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway for counteracting cytotoxic and mutagenic effects of UV irradiation. To provide insight into the in vivo regulation of the DNA damage recognition step of global genome NER (GG-NER), we constructed cell lines expressing fluorescently tagged damaged DNA binding protein 1 (DDB1). DDB1 is a core subunit of a number of cullin 4-RING ubiquitin ligase complexes. UV-activated DDB1-DDB2-CUL4A-ROC1 ubiquitin ligase participates in the initiation of GG-NER and triggers the UV-dependent degradation of its subunit DDB2. We found that DDB1 rapidly accumulates on DNA damage sites. However, its binding to damaged DNA is not static, since DDB1 constantly dissociates from and binds to DNA lesions. DDB2, but not CUL4A, was indispensable for binding of DDB1 to DNA damage sites. The residence time of DDB1 on the damage site is independent of the main damage-recognizing protein of GG-NER, XPC, as well as of UV-induced proteolysis of DDB2. The amount of DDB1 that is temporally immobilized on damaged DNA critically depends on DDB2 levels in the cell. We propose a model in which UV-dependent degradation of DDB2 is important for the release of DDB1 from continuous association to unrepaired DNA and makes DDB1 available for its other DNA damage response functions.     

Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles.     

HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP)--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP) E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.     

PCNA-coupled p21 degradation after DNA damage: The exception that confirms the rule?

While many are the examples of DNA damaging treatments that induce p21 accumulation, the conception of p21 upregulation as the universal response to genotoxic stress has come to an end. Compelling evidences have demonstrated the existence of converging signals that negatively regulate p21 bellow basal levels when replication forks are blocked. Moreover, conclusive reports identified the E3-ligase CRL4^CDT2 (CUL4–DDB1–CDT2) as the enzymatic complex that promotes p21 proteolysis when treatments such as UV irradiation trigger replication fork stress. A pre-requisite for CRL4^CDT2-driven proteolysis is the interaction of p21 with PCNA. Interestingly as well, CRL4^CDT2-dependent proteolysis is not limited to p21 and affects other PCNA partners, including the specialized DNA polymerase η (pol eta). These recent discoveries are particularly intriguing since the UV-induced degradation of p21 has been shown to be required for efficient pol η recruitment to DNA lesions. Herein we review the findings that lead to the identification of the molecular mechanism that triggers damage-induced PCNA-coupled protein proteolysis. We propose a novel model in which CRL4^CDT2-dependent protein degradation facilitates a sequential and dynamic exchange between PIP box bearing proteins at stall forks during Translesion DNA synthesis (TLS). Moreover, given the tight spatiotemporal control that CRL4^CDT2-driven proteolysis is able to confer to PCNA-regulated processes, we discuss the impact that this degradation mechanism might have in other molecular switches associated with the repair of damaged DNA.     

Opposing effects of glutamine and asparagine govern prion formation by intrinsically disordered proteins

Several proteins, including the replication licensing factor CDT1 and the histone methyltransferase SET8, are targeted for proteolysis during DNA replication and repair by the E3 ubiquitin ligase CRL4(CDT2). CRL4(CDT2) function is coupled to replication and repair because it only ubiquitinates substrates that associate with chromatin-bound PCNA. Here, we report a genome-wide siRNA screen that identifies multiple factors necessary for CDT1 destruction after UV irradiation. Among these, nucleotide excision repair factors promote CDT1 destruction due to a role in recruiting PCNA to damaged DNA. The COP9/Signalosome regulates CDT2 stability through CUL4 deneddylation. Finally, the p97 AAA(+)-ATPase and its cofactor UFD1 are required for proteasome-dependent removal of ubiquitinated CDT1 and SET8 from chromatin and their subsequent degradation both in vivo and in a Xenopus egg extract system in vitro. This study provides insight into and a resource for the further exploration of pathways that promote timely degradation of chromatin-associated CRL4(CDT2) substrates.     

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.     

Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis

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.     

CRL4-DDB1-VPRBP ubiquitin ligase mediates the stress triggered proteolysis of Mcm10

When mammalian cells experience radiation insult, DNA replication is stalled to prevent erroneous DNA synthesis. UV-irradiation triggers proteolysis of Mcm10, an essential human replication factor, inhibiting the ongoing replication. Here, we report that Mcm10 associates with E3 ubiquitin ligase comprising DNA damage-binding protein, DDB1, cullin, Cul4 and ring finger protein, Roc1. Depletion of DDB1, Roc1 or Cul4 abrogates the UV-triggered Mcm10 proteolysis, implying that Cul4-Roc1-DDB1 ubiquitin ligase mediates Mcm10 downregulation. The purified Cul4-Roc1-DDB1 complex ubiquitinates Mcm10 in vitro, proving that Mcm10 is its substrate. By screening the known DDB1 interacting proteins, we discovered that VprBP is the substrate recognition subunit that targets Mcm10 for degradation. Hence, these results establish that Cul4-DDB1-VprBP ubiquitin ligase mediates the stress-induced proteolysis of replication factor, Mcm10.     

Rtt101 and Mms1 in budding yeast form a CUL4(DDB1)-like ubiquitin ligase that promotes replication through damaged DNA

In budding yeast the cullin Rtt101 promotes replication fork progression through natural pause sites and areas of DNA damage, but its relevant subunits and molecular mechanism remain poorly understood. Here, we show that in budding yeast Mms1 and Mms22 are functional subunits of an Rtt101-based ubiquitin ligase that associates with the conjugating-enzyme Cdc34. Replication forks in mms1Delta, mms22Delta and rtt101Delta cells are sensitive to collisions with drug-induced DNA lesions, but not to transient pausing induced by nucleotide depletion. Interaction studies and sequence analysis have shown that Mms1 resembles human DDB1, suggesting that Rtt101(Mms1) is the budding yeast counterpart of the mammalian CUL4(DDB1) ubiquitin ligase family. Rtt101 interacts in an Mms1-dependent manner with the putative substrate-specific adaptors Mms22 and Crt10, the latter being a regulator of expression of ribonucleotide reductase. Taken together, our data suggest that the Rtt101(Mms1) ubiquitin ligase complex might be required to reorganize replication forks that encounter DNA lesions.     

HIV-1 Vpr Loads Uracil DNA Glycosylase-2 onto DCAF1, a Substrate Recognition Subunit of a Cullin 4A-RING E3 Ubiquitin Ligase for Proteasome-dependent Degradation

The human immunodeficiency virus type 1 (HIV-1) accessory protein, Vpr, interacts with several host cellular proteins including uracil DNA glycosylase-2 (UNG2) and a cullin-RING E3 ubiquitin ligase assembly (CRL4^DCAF1). The ligase is composed of cullin 4A (CUL4A), RING H2 finger protein (RBX1), DNA damage-binding protein 1 (DDB1), and a substrate recognition subunit, DDB1- and CUL4-associated factor 1 (DCAF1). Here we show that recombinant UNG2 specifically interacts with Vpr, but not with Vpx of simian immunodeficiency virus, forming a heterotrimeric complex with DCAF1 and Vpr in vitro as well as in vivo. Using reconstituted CRL4^DCAF1 and CRL4^DCAF1-Vpr E3 ubiquitin ligases in vitro reveals that UNG2 ubiquitination (ubiquitylation) is facilitated by Vpr. Co-expression of DCAF1 and Vpr causes down-regulation of UNG2 in a proteasome-dependent manner, with Vpr mutants that are defective in UNG2 or DCAF1 binding abrogating this effect. Taken together, our results show that the CRL4^DCAF1 E3 ubiquitin ligase can be subverted by Vpr to target UNG2 for degradation.     

The <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>CDT-2 ubiquitin ligase is required for attenuation of EGFR signalling in vulva precursor cells

Background  

Attenuation of the EGFR (Epidermal Growth Factor Receptor) signalling cascade is crucial to control cell fate during development. A candidate-based RNAi approach in C. elegans identified CDT-2 as an attenuator of LET-23 (EGFR) signalling. Human CDT2 is a component of the conserved CDT2/CUL4/DDB1 ubiquitin ligase complex that plays a critical role in DNA replication and G2/M checkpoint. Within this complex, CDT2 is responsible for substrate recognition. This ubiquitin ligase complex has been shown in various organisms, including C. elegans, to target the replication-licensing factor CDT1, and the CDK inhibitor p21. However, no previous link to EGFR signalling has been identified.     

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.     

CRL4CDT2 Targets CHK1 for PCNA-Independent Destruction

CDT2 targets proteins involved in replication licensing (CDT1), cell cycle control (p21), and chromatin modification (SET8) for destruction by the CUL4-based E3 ligase (CRL4). CRL4^CDT2 recruits these substrates through interactions with chromatin-bound PCNA and ubiquitinates them exclusively on chromatin. Rereplication and G₂ cell cycle arrest are observed in CDT2-depleted cells. The rereplication phenotype has been attributed to an inability to destroy CDT1, but the molecular target important for G₂ cell cycle arrest in CDT2-depleted cells has not been identified. Here we identify CHK1 as a novel CRL4^CDT2 substrate and demonstrate that CHK1 activity is required for maintaining G₂ arrest in CDT2-depleted cells. We demonstrate that CRL4^CDT2 targets the activated form of CHK1 for destruction in the nucleoplasm rather than on chromatin and that this occurs in a PCNA-independent manner. Although both CRL1 and CRL4 ubiquitinate CHK1, we report that they bind CHK1 in distinct cellular compartments. Our study provides insight into how elevated CDT2 expression levels may provide tumors with a proliferative advantage.