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.     

Ribonucleotide reductase activity is coupled to DNA synthesis via proliferating cell nuclear antigen

Synthesis of deoxynucleoside triphosphates (dNTPs) is required for both DNA replication and DNA repair and is catalyzed by ribonucleotide reductases (RNR), which convert ribonucleotides to their deoxy forms [1, 2]. Maintaining the correct levels of dNTPs for DNA synthesis is important for minimizing the mutation rate [3-7], and this is achieved by tight regulation of RNR [2, 8, 9]. In fission yeast, RNR is regulated in part by a small protein inhibitor, Spd1, which is degraded in S phase and after DNA damage to allow upregulation of dNTP supply [10-12]. Spd1 degradation is mediated by the activity of the CRL4(Cdt2) ubiquitin ligase complex [5, 13, 14]. This has been reported to be dependent on modulation of Cdt2 levels, which are cell cycle regulated, peaking in S phase, and which also increase after DNA damage in a checkpoint-dependent manner [7, 13]. We show here that Cdt2 level fluctuations are not sufficient to regulate Spd1 proteolysis and that the key step in this event is the interaction of Spd1 with the polymerase processivity factor proliferating cell nuclear antigen (PCNA), complexed onto DNA. This mechanism thus provides a direct link between DNA synthesis and RNR regulation.     

A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1

Cul4 E3 ubiquitin ligases contain the cullin 4 scaffold and the triple beta propeller Ddb1 adaptor protein, but few substrate receptors have been identified. Here, we identify 18 Ddb1- and Cul4-associated factors (DCAFs), including 14 containing WD40 repeats. DCAFs interact with multiple surfaces on Ddb1, and the interaction of WD40-containing DCAFs with Ddb1 requires a conserved "WDXR" motif. DCAF2/Cdt2, which is related to S. pombe Cdt2, functions in Xenopus egg extracts and human cells to destroy the replication licensing protein Cdt1 in S phase and after DNA damage. Depletion of human Cdt2 causes rereplication and checkpoint activation. In Xenopus, Cdt2 is recruited to replication forks via Cdt1 and PCNA, where Cdt1 ubiquitylation occurs. These studies uncover diverse substrate receptors for Cul4 and identify Cdt2 as a conserved component of the Cul4-Ddb1 E3 that is essential to destroy Cdt1 and ensure proper cell cycle regulation of DNA replication.     

COP9 signalosome: a provider of DNA building blocks

In fission yeast, the COP9 signalosome is required to activate ribonucleotide reductase for DNA synthesis. This is mediated via the ubiquitin ligase Pcu4, activation of which leads to degradation of the scaffold protein Spd1, which anchors the small ribonucleotide reductase subunit in the nucleus away from the large subunit in the cytoplasm.     

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.     

DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1

Cdt1 is an essential protein required for licensing of replication origins. Here, we show that in Schizosaccharomyces pombe, Cdt1 is proteolysed in M and G1 phases in response to DNA damage and that this mechanism seems to be conserved from yeast to Metazoa. This degradation does not require Rad3 and Cds1, indicating that it is independent of classic DNA damage and replication checkpoint pathways. Damage-induced degradation of Cdt1 is dependent on Cdt2 and Ddb1, which are components of a Cul4 ubiquitin ligase. We also show that Cdt2 and Ddb1 are needed for cell-cycle changes in Cdt1 levels in the absence of DNA damage. Cdt2 and Ddb1 have been shown to be involved in the degradation of the Spd1 inhibitor of ribonucleotide reductase after DNA damage, and we speculate that Cdt1 downregulation might contribute to genome stability by reducing demand on dNTP pools during DNA repair.     

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.     

Selective ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G ubiquitin-conjugating enzymes via the CRL4Cdt2 ubiquitin ligase complex

CRL4(Cdt2) is a cullin-based E3 ubiquitin ligase that promotes the ubiquitin-dependent proteolysis of various substrates implicated in the control of cell cycle and various DNA metabolic processes such as DNA replication and repair. Substrates for CRL4(Cdt2) E3 ubiquitin ligase include the replication licensing factor Cdt1 and the cyclin-dependent kinase (Cdk) inhibitor p21. Inhibition of this E3 ligase leads to serious abnormalities of the cell cycle and cell death. The ubiquitin-conjugating enzyme (UBC) involved in this important pathway, however, remains unknown. By a proteomic analysis of Cdt2-associated proteins and an RNA interference-based screening approach, we show that CRL4(Cdt2) utilizes two different UBCs to target different substrates. UBCH8, a member of the UBE2E family of UBCs, ubiquitylates and promotes the degradation of p21, both during the normal cell cycle and in UV-irradiated cells. Importantly, depletion of UBCH8 by small interfering RNA (siRNA) increases p21 protein level, delays entry into S phase of the cell cycle, and suppresses the DNA damage response after UV irradiation. On the other hand, members of the UBE2G family of UBCs (UBE2G1 and UBE2G2) cooperate with CRL4(Cdt2) to polyubiquitylate and degrade Cdt1 postradiation, an activity that is critical for preventing origin licensing in DNA-damaged cells. Finally, we show that UBCH8, but not UBE2G1 or UBE2G2, is required for CRL4(Cdt2)-mediated ubiquitylation and degradation of the histone H4 lysine 20 monomethyltransferase Set8, a previously identified CRL4(Cdt2) substrate, as well as for CRL4(Cdt2)-dependent monoubiquitylation of PCNA in unstressed cells. These findings identify the UBCs required for the activity of CRL4(Cdt2) on multiple substrates and demonstrate that different UBCs are involved in the selective ubiquitylation of different substrates by the same E3 complex.     

Mitotic UV Irradiation Induces a DNA Replication-Licensing Defect that Potentiates G1 Arrest Response

Cdt1 begins to accumulate in M phase and has a key role in establishing replication licensing at the end of mitosis or in early G1 phase. Treatments that damage the DNA of cells, such as UV irradiation, induce Cdt1 degradation through PCNA-dependent CRL4-Cdt2 ubiquitin ligase. How Cdt1 degradation is linked to cell cycle progression, however, remains unclear. In G1 phase, when licensing is established, UV irradiation leads to Cdt1 degradation, but has little effect on the licensing state. In M phase, however, UV irradiation does not induce Cdt1 degradation. When mitotic UV-irradiated cells were released into G1 phase, Cdt1 was degraded before licensing was established. Thus, these cells exhibited both defective licensing and G1 cell cycle arrest. The frequency of G1 arrest increased in cells expressing extra copies of Cdt2, and thus in cells in which Cdt1 degradation was enhanced, whereas the frequency of G1 arrest was reduced in cell expressing an extra copy of Cdt1. The G1 arrest response of cells irradiated in mitosis was important for cell survival by preventing the induction of apoptosis. Based on these observations, we propose that mammalian cells have a DNA replication-licensing checkpoint response to DNA damage induced during mitosis.     

Proliferating cell nuclear antigen-dependent rapid recruitment of Cdt1 and CRL4Cdt2 at DNA-damaged sites after UV irradiation in HeLa cells

The licensing factor Cdt1 is degraded by CRL4(Cdt2) ubiquitin ligase dependent on proliferating cell nuclear antigen (PCNA) during S phase and when DNA damage is induced in G(1) phase. Association of both Cdt2 and PCNA with chromatin was observed in S phase and after UV irradiation. Here we used a micropore UV irradiation assay to examine Cdt2 accumulation at cyclobutane pyrimidine dimer-containing DNA-damaged sites in the process of Cdt1 degradation in HeLa cells. Cdt2, present in the nucleus throughout the cell cycle, accumulated rapidly at damaged DNA sites during G(1) phase. The recruitment of Cdt2 is dependent on prior PCNA chromatin binding because Cdt2 association was prevented when PCNA was silenced. Cdt1 was also recruited to damaged sites soon after UV irradiation through its PIP-box. As Cdt1 was degraded, the Cdt2 signal at damaged sites was reduced, but PCNA, cyclobutane pyrimidine dimer, and XPA (xeroderma pigmentosum, complementation group A) signals remained at the same levels. These findings suggest that Cdt1 degradation following UV irradiation occurs rapidly at damaged sites due to PCNA chromatin loading and the recruitment of Cdt1 and CRL4(Cdt2), before DNA damage repair is completed.     

Cdt1 and Cdc6 are destabilized by rereplication-induced DNA damage

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.     

Mismatch repair proteins recruited to ultraviolet light-damaged sites lead to degradation of licensing factor Cdt1 in the G1 phase

Cdt1 is rapidly degraded by CRL4^Cdt2 E3 ubiquitin ligase after UV (UV) irradiation. Previous reports revealed that the nucleotide excision repair (NER) pathway is responsible for the rapid Cdt1-proteolysis. Here, we show that mismatch repair (MMR) proteins are also involved in the degradation of Cdt1 after UV irradiation in the G1 phase. First, compared with the rapid (within ～15 min) degradation of Cdt1 in normal fibroblasts, Cdt1 remained stable for ～30 min in NER-deficient XP-A cells, but was degraded within ～60 min. The delayed degradation was also dependent on PCNA and CRL4^Cdt2. The MMR proteins Msh2 and Msh6 were recruited to the UV-damaged sites of XP-A cells in the G1 phase. Depletion of these factors with small interfering RNAs prevented Cdt1 degradation in XP-A cells. Similar to the findings in XP-A cells, depletion of XPA delayed Cdt1 degradation in normal fibroblasts and U2OS cells, and co-depletion of Msh6 further prevented Cdt1 degradation. Furthermore, depletion of Msh6 alone delayed Cdt1 degradation in both cell types. When Cdt1 degradation was attenuated by high Cdt1 expression, repair synthesis at the damaged sites was inhibited. Our findings demonstrate that UV irradiation induces multiple repair pathways that activate CRL4^Cdt2 to degrade its target proteins in the G1 phase of the cell cycle, leading to efficient repair of DNA damage.     

The Cul4-Ddb1(Cdt)² ubiquitin ligase inhibits invasion of a boundary-associated antisilencing factor into heterochromatin

Partitioning of chromosomes into euchromatic and heterochromatic domains requires mechanisms that specify boundaries. The S. pombe JmjC family protein Epe1 prevents the ectopic spread of heterochromatin and is itself concentrated at boundaries. Paradoxically, Epe1 is recruited to heterochromatin by HP1 silencing factors that are distributed throughout heterochromatin. We demonstrate here that the selective enrichment of Epe1 at boundaries requires its regulation by the conserved Cul4-Ddb1(Cdt)2 ubiquitin ligase, which directly recognizes Epe1 and promotes its polyubiquitylation and degradation. Strikingly, in cells lacking the ligase, Epe1 persists in the body of heterochromatin thereby inducing a defect in gene silencing. Epe1 is the sole target of the Cul4-Ddb1(Cdt)2 complex whose destruction is necessary for the preservation of heterochromatin. This mechanism acts parallel with phosphorylation of HP1/Swi6 by CK2 to restrict Epe1. We conclude that the ubiquitin-dependent sculpting of the chromosomal distribution of an antisilencing factor is critical for heterochromatin boundaries to form correctly.     

Checkpoint Kinase ATR Phosphorylates Cdt2, a Substrate Receptor of CRL4 Ubiquitin Ligase,and Promotes the Degradation of Cdt1 following UV Irradiation

The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4^Cdt2 for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.     

CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase

The proper coordination between DNA replication and mitosis during cell-cycle progression is crucial for genomic stability. During G2 and mitosis, Set8 catalyzes monomethylation of histone H4 on lysine 20 (H4K20me1), which promotes chromatin compaction. Set8 levels decline in S phase, but why and how this occurs is unclear. Here, we show that Set8 is targeted for proteolysis in S phase and in response to DNA damage by the E3 ubiquitin ligase, CRL4(Cdt2). Set8 ubiquitylation occurs on chromatin and is coupled to DNA replication via a specific degron in Set8 that binds PCNA. Inactivation of CRL4(Cdt2) leads to Set8 stabilization and aberrant H4K20me1 accumulation in replicating cells. Transient S phase expression of a Set8 mutant lacking the degron promotes premature H4K20me1 accumulation and chromatin compaction, and triggers a checkpoint-mediated G2 arrest. Thus, CRL4(Cdt2)-dependent destruction of Set8 in S phase preserves genome stability by preventing aberrant chromatin compaction during DNA synthesis.     

Regulation of TGF-β signaling,exit from the cell cycle,and cellular migration through cullin cross-regulation: SCF-FBXO11 turns off CRL4-Cdt2

Deregulation of the cell cycle and genome instability are common features of cancer cells and various mechanisms exist to preserve the integrity of the genome and guard against cancer. The cullin 4-RING ubiquitin ligase (CRL4) with the substrate receptor Cdt2 (CRL4^Cdt2) promotes cell cycle progression and prevents genome instability through ubiquitylation and degradation of Cdt1, p21, and Set8 during S phase of the cell cycle and following DNA damage. Two recently published studies report the ubiquitin-dependent degradation of Cdt2 via the cullin 1-RING ubiquitin ligase (CRL1) in association with the substrate specificity factor and tumor suppressor FBXO11 (CRL1^FBXO11). The newly identified pathway restrains the activity of CRL4^Cdt2 on p21 and Set8 and regulates cellular response to TGF-β, exit from the cell cycle and cellular migration. Here, we show that the CRL1^FBXO11 also promotes the degradation of Cdt2 during an unperturbed cell cycle to promote efficient progression through S and G₂/M phases of the cell cycle. We discuss how this new method of regulating the abundance of Cdt2 participates in various cellular activities.     

Cdt2-mediated XPG degradation promotes gap-filling DNA synthesis in nucleotide excision repair

Xeroderma pigmentosum group G (XPG) protein is a structure-specific repair endonuclease, which cleaves DNA strands on the 3′ side of the DNA damage during nucleotide excision repair (NER). XPG also plays a crucial role in initiating DNA repair synthesis through recruitment of PCNA to the repair sites. However, the fate of XPG protein subsequent to the excision of DNA damage has remained unresolved. Here, we show that XPG, following its action on bulky lesions resulting from exposures to UV irradiation and cisplatin, is subjected to proteasome-mediated proteolytic degradation. Productive NER processing is required for XPG degradation as both UV and cisplatin treatment-induced XPG degradation is compromised in NER-deficient XP-A, XP-B, XP-C, and XP-F cells. In addition, the NER-related XPG degradation requires Cdt2, a component of an E3 ubiquitin ligase, CRL4^Cdt2. Micropore local UV irradiation and in situ Proximity Ligation assays demonstrated that Cdt2 is recruited to the UV-damage sites and interacts with XPG in the presence of PCNA. Importantly, Cdt2-mediated XPG degradation is crucial to the subsequent recruitment of DNA polymerase δ and DNA repair synthesis. Collectively, our data support the idea of PCNA recruitment to damage sites which occurs in conjunction with XPG, recognition of the PCNA-bound XPG by CRL4^Cdt2 for specific ubiquitylation and finally the protein degradation. In essence, XPG elimination from DNA damage sites clears the chromatin space needed for the subsequent recruitment of DNA polymerase δ to the damage site and completion of gap-filling DNA synthesis during the final stage of NER.     

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.