A damaged DNA binding protein 2 mutation disrupting interaction with proliferating-cell nuclear antigen affects DNA repair and confers proliferation advantage

In mammalian cells, Nucleotide Excision Repair (NER) plays a role in removing DNA damage induced by UV radiation. In Global Genome-NER subpathway, DDB2 protein forms a complex with DDB1 (UV-DDB), recognizing photolesions. During DNA repair, DDB2 interacts directly with PCNA through a conserved region in N-terminal tail and this interaction is important for DDB2 degradation. In this work, we sought to investigate the role of DDB2-PCNA association in DNA repair and cell proliferation after UV-induced DNA damage. To this end, stable clones expressing DDB2^Wt and DDB2^PCNA- were used. We have found that cells expressing a mutant DDB2 show inefficient photolesions removal, and a concomitant lack of binding to damaged DNA in vitro. Unexpected cellular behaviour after DNA damage, such as UV-resistance, increased cell growth and motility were found in DDB2^PCNA- stable cell clones, in which the most significant defects in cell cycle checkpoint were observed, suggesting a role in the new cellular phenotype. Based on these findings, we propose that DDB2-PCNA interaction may contribute to a correct DNA damage response for maintaining genome integrity.     

DDB2 association with PCNA is required for its degradation after UV-induced DNA damage

DDB2 is a protein playing an essential role in the lesion recognition step of the global genome sub-pathway of nucleotide excision repair (GG-NER) process. Among the proteins involved in the DNA damage response, p21^CDKN1A (p21) has been reported to participate in NER, but also to be removed by proteolytic degradation, thanks to its association with PCNA. DDB2 is involved in the CUL4-DDB1 complex mediating p21 degradation; however, the direct interaction between DDB2, p21 and PCNA has been never investigated. Here, we show that DDB2 co-localizes with PCNA and p21 at local UV-induced DNA-damage sites, and these proteins co-immunoprecipitate in the same complex. In addition, we provide evidence that p21 is not able to bind directly DDB2, but, to this end, the presence of PCNA is required. Direct physical association of recombinant DDB2 protein with PCNA is mediated by a conserved PIP-box present in the N-terminal region of DDB2. Mutation of the PIP-box resulted in the loss of protein interaction. Interestingly, the same mutation, or depletion of PCNA by RNA interference, greatly impaired DDB2 degradation induced by UV irradiation. These results indicate that DDB2 is a PCNA-binding protein, and that this association is required for DDB2 proteolytic degradation.     

The xeroderma pigmentosum group E gene product DDB2 activates nucleotide excision repair by regulating the level of p21Waf1/Cip1

The xeroderma pigmentosum group E gene product DDB2, a protein involved in nucleotide excision repair (NER), associates with the E3 ubiquitin ligase complex Cul4A-DDB1. But the precise role of these interactions in the NER activity of DDB2 is unclear. Several models, including DDB2-mediated ubiquitination of histones in UV-irradiated cells, have been proposed. But those models lack clear genetic evidence. Here we show that DDB2 participates in NER by regulating the cellular levels of p21^Waf1/Cip1. We show that DDB2 enhances nuclear accumulation of DDB1, which binds to a modified form of p53 containing phosphorylation at Ser18 (p53^S18P) and targets it for degradation in low-dose-UV-irradiated cells. DDB2^−/− mouse embryonic fibroblasts (MEFs), unlike wild-type MEFs, are deficient in the proteolysis of p53^S18P. Accumulation of p53^S18P in DDB2^−/− MEFs causes higher expression p21^Waf1/Cip1. We show that the increased expression of p21^Waf1/Cip1 is the cause NER deficiency in DDB2^−/− cells because deletion or knockdown of p21^Waf1/Cip1 reverses their NER-deficient phenotype. p21^Waf1/Cip1 was shown to bind PCNA, which is required for both DNA replication and NER. Moreover, an increased level of p21^Waf1/Cip1 was shown to inhibit NER both in vitro and in vivo. Our results provide genetic evidence linking the regulation of p21^Waf1/Cip1 to the NER activity of DDB2.     

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21^CDKN1A, thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3′-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21^−/− fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA–p21 dependent manner.     

Nucleotide Excision Repair Is Associated with the Replisome and Its Efficiency Depends on a Direct Interaction between XPA and PCNA

Proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication, DNA repair, cell cycle regulation, chromatin remodeling, and epigenetics. Many proteins interact with PCNA through the PCNA interacting peptide (PIP)-box or the newly identified AlkB homolog 2 PCNA interacting motif (APIM). The xeroderma pigmentosum group A (XPA) protein, with a central but somewhat elusive role in nucleotide excision repair (NER), contains the APIM sequence suggesting an interaction with PCNA. With an in vivo based approach, using modern techniques in live human cells, we show that APIM in XPA is a functional PCNA interacting motif and that efficient NER of UV lesions is dependent on an intact APIM sequence in XPA. We show that XPA^−/− cells complemented with XPA containing a mutated APIM sequence have increased UV sensitivity, reduced repair of cyclobutane pyrimidine dimers and (6–4) photoproducts, and are consequently more arrested in S phase as compared to XPA^−/− cells complemented with wild type XPA. Notably, XPA colocalizes with PCNA in replication foci and is loaded on newly synthesized DNA in undamaged cells. In addition, the TFIIH subunit XPD, as well as XPF are loaded on DNA together with XPA, and XPC and XPG colocalize with PCNA in replication foci. Altogether, our results suggest a presence of the NER complex in the vicinity of the replisome and a novel role of NER in post-replicative repair.     

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.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

Role of Claspin in regulation of nucleotide excision repair factor DDB2

The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.     

DDB2 (Damaged-DNA binding protein 2) in nucleotide excision repair and DNA damage response

DDB2 was identified as a protein involved in the Nucleotide Excision Repair (NER), a major DNA repair mechanism that repairs UV damage to prevent accumulation of mutations and tumorigenesis. However, recent studies indicated additional functions of DDB2 in the DNA damage response pathway. Herein, we discuss the proposed mechanisms by which DDB2 activates NER and programmed cell death upon DNA damage through its E3 ligase activity.     

Physical and functional interaction between DDB and XPA in nucleotide excision repair

Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction.     

Regulation of Nucleotide Excision Repair by Nuclear Lamin B1

The nuclear lamins play important roles in the structural organization and function of the metazoan cell nucleus. Recent studies on B-type lamins identified a requirement for lamin B1 (LB1) in the regulation of cell proliferation in normal diploid cells. In order to further investigate the function of LB1 in proliferation, we disrupted its normal expression in U-2 OS human osteosarcoma and other tumor cell lines. Silencing LB1 expression induced G1 cell cycle arrest without significant apoptosis. The arrested cells are unable to mount a timely and effective response to DNA damage induced by UV irradiation. Several proteins involved in the detection and repair of UV damage by the nucleotide excision repair (NER) pathway are down-regulated in LB1 silenced cells including DDB1, CSB and PCNA. We propose that LB1 regulates the DNA damage response to UV irradiation by modulating the expression of specific genes and activating persistent DNA damage signaling. Our findings are relevant to understanding the relationship between the loss of LB1 expression, DNA damage signaling, and replicative senescence.     

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.     

Artemis interacts with the Cul4A-DDB1DDB2 ubiquitin E3 ligase and regulates degradation of the CDK inhibitor p27

Artemis, a member of the SNM1 gene family, is a multifunctional phospho-protein that has been shown to have important roles in V(D)J recombination, DNA double-strand break repair and stress-induced cell cycle checkpoint regulation. We show here that Artemis interacts with the Cul4A-DDB1 E3 ubiquitin ligase via a direct interaction with the substrate-specificity receptor DDB2. Furthermore, Artemis also interacts with the CDK inhibitor and tumor suppressor p27, a substrate of the Cul4A-DDB1 ligase, and both DDB2 and Artemis are required for the degradation of p27 mediated by this complex. We also show that the regulation of p27 by Artemis and DDB2 is important for cell cycle progression in normally proliferating cells and in response to serum deprivation. These findings thus define a function for Artemis as an effector of Cullin-based E3 ligase-mediated ubiquitylation, demonstrate a novel pathway for the regulation of p27 and show that Cul4A-DDB1^DDB2-Artemis regulates G₁-phase cell cycle progression in mammalian cells.Key words: artemis, DDB2, p27, Cul4A-DDB1, ubiquitylation     

Detection of bulky DNA lesions: DDB2 at the interface of chromatin and DNA repair in eukaryotes

Bulky DNA damage is corrected by the nucleotide excision repair (NER) pathway. Although the core biochemical mechanism of NER is understood, details including lesion recognition and repair in the context of chromatin remain to be elucidated. As more data become available, the complexity of lesion recognition in chromatin is becoming clear. This review will discuss current knowledge of DNA damage recognition in the context of chromatin, with a focus on the roles of chromatin remodeling and the specific lesion recognition protein DDB2 (DNA damage-binding protein 2) in chromatin repair. Additionally, we propose a model that ubiquitination-mediated DDB2 dissociation from chromatin, not its degradation, is important for GG-NER progression.     

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.     

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.     

L2DTL/CDT2 Interacts with the CUL4/DDB1 Complex and PCNA and Regulates CDT1 Proteolysis in Response to DNA Damage

The CUL4 (cullin 4) proteins are the core components of a new class of ubiquitin E3 ligases that regulate cell cycle, DNA replication, and DNA damage response. To determine the composition of CUL4 ubiquitin E3 ligase complex, we used anti-CUL4 antibody affinity chromatography to isolate the proteins that associated with human CUL4 complexes and identified them by mass-spectrometry. A novel and conserved WD40 domain-containing protein, the human homologue of Drosophila lethal(2) denticleless protein (L2DTL), was found to associate with CUL4 and DDB1. L2DTL also interacts with replication licensing protein CDT1 in vivo. Loss of L2DTL in Drosophila S2 and human cells suppressed proteolysis of CDT1 in response to DNA damage. We further isolated the human L2DTL complexes by anti-L2DTL immuno-affinity chromatography from HeLa cells and found it associates with DDB1, components of the COP9-signalosome complex (CSN), and PCNA. We found that PCNA interacts with CDT1 and loss of PCNA suppressed CDT1 proteolysis after DNA damage. Our data also revealed that in vivo, inactivation of L2DTL causes the dissociation of DDB1 from the CUL4 complex. Our studies suggest that L2DTL and PCNA interact with CUL4/DDB1 complexes and are involved in CDT1 degradation after DNA damage.