Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.     

DNA repair factor XPC is modified by SUMO-1 and ubiquitin following UV irradiation

Nucleotide excision repair (NER) is the major DNA repair process that removes diverse DNA lesions including UV-induced photoproducts. There are more than 20 proteins involved in NER. Among them, XPC is thought to be one of the first proteins to recognize DNA damage during global genomic repair (GGR), a sub-pathway of NER. In order to study the mechanism through which XPC participates in GGR, we investigated the possible modifications of XPC protein upon UV irradiation in mammalian cells. Western blot analysis of cell lysates from UV-irradiated normal human fibroblast, prepared by direct boiling in an SDS lysis buffer, showed several anti-XPC antibody-reactive bands with molecular weight higher than the original XPC protein. The reciprocal immunoprecipitation and siRNA transfection analysis demonstrated that XPC protein is modified by SUMO-1 and ubiquitin. By using several NER-deficient cell lines, we found that DDB2 and XPA are required for UV-induced XPC modifications. Interestingly, both the inactivation of ubiquitylation and the treatment of proteasome inhibitors quantitatively inhibited the UV-induced XPC modifications. Furthermore, XPC protein is degraded significantly following UV irradiation in XP-A cells in which sumoylation of XPC does not occur. Taken together, we conclude that XPC protein is modified by SUMO-1 and ubiquitin following UV irradiation and these modifications require the functions of DDB2 and XPA, as well as the ubiquitin–proteasome system. Our results also suggest that at least one function of UV-induced XPC sumoylation is related to the stabilization of XPC protein.     

UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells

Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.     

The p38 mitogen-activated protein kinase augments nucleotide excision repair by mediating DDB2 degradation and chromatin relaxation

The p38 MAPK is a family of serine/threonine protein kinases that play important roles in cellular responses to external stress signals, e.g. UV irradiation. To assess the role of p38 MAPK pathway in nucleotide excision repair (NER), the most versatile DNA repair pathway, we determined the efficiency of NER in cells treated with p38 MAPK inhibitor SB203580 and found that p38 MAPK is required for the prompt repair of UV-induced DNA damage CPD. We further investigated the possible mechanism through which p38 MAPK regulates NER and found that p38 MAPK mediates UV-induced histone H3 acetylation and chromatin relaxation. Moreover, p38 MAPK also regulates UV-induced DDB2 ubiquitylation and degradation via phosphorylation of the target protein. Finally, our results showed that p38 MAPK is required for the recruitment of NER factors XPC and TFIIH to UV-induced DNA damage sites. We conclude that p38 MAPK regulates chromatin remodeling as well as DDB2 degradation for facilitating NER factor assembly.     

DDB2-Independent Role for p53 in the Recovery from Ultraviolet Light-Induced Replication Arrest

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion DNA synthesis of these DNA lesions.     

Xeroderma pigmentosum group A protein loads as a separate factor onto DNA lesions

Nucleotide excision repair (NER) is the main DNA repair pathway in mammals for removal of UV-induced lesions. NER involves the concerted action of more than 25 polypeptides in a coordinated fashion. The xeroderma pigmentosum group A protein (XPA) has been suggested to function as a central organizer and damage verifier in NER. How XPA reaches DNA lesions and how the protein is distributed in time and space in living cells are unknown. Here we studied XPA in vivo by using a cell line stably expressing physiological levels of functional XPA fused to green fluorescent protein and by applying quantitative fluorescence microscopy. The majority of XPA moves rapidly through the nucleoplasm with a diffusion rate different from those of other NER factors tested, arguing against a preassembled XPA-containing NER complex. DNA damage induced a transient ( approximately 5-min) immobilization of maximally 30% of XPA. Immobilization depends on XPC, indicating that XPA is not the initial lesion recognition protein in vivo. Moreover, loading of replication protein A on NER lesions was not dependent on XPA. Thus, XPA participates in NER by incorporation of free diffusing molecules in XPC-dependent NER-DNA complexes. This study supports a model for a rapid consecutive assembly of free NER factors, and a relatively slow simultaneous disassembly, after repair.     

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.     

Telomerase-immortalized human fibroblasts retain UV-induced mutagenesis and p53-mediated DNA damage responses

Immortalized cells frequently have disruptions of p53 activity and lack p53-dependent nucleotide excision repair (NER). We hypothesized that telomerase immortalization would not alter p53-mediated ultraviolet light (UV)-induced DNA damage responses. DNA repair proficient primary diploid human fibroblasts (GM00024) were immortalized by transduction with a telomerase expressing retrovirus. Empty retrovirus transduced cells senesced after a few doublings. Telomerase transduced GM00024 cells (tGM24) were cultured continuously for 6 months (>60 doublings). Colony forming ability after UV irradiation was dose-dependent between 0 and 20J/m2 UVC (LD50=5.6J/m2). p53 accumulation was UV dose- and time-dependent as was induction of p48(XPE/DDB2), p21(CIP1/WAF1), and phosphorylation on p53-S15. UV dose-dependent apoptosis was measured by nuclear condensation. UV exposure induced UV-damaged DNA binding as monitored by electrophoretic mobility shift assays using UV irradiated radiolabeled DNA probe was inhibited by p53-specific siRNA transfection. p53-Specific siRNA transfection also prevented UV induction of p48 and improved UV survival measured by colony forming ability. Strand-specific NER of cyclobutane pyrimidine dimers (CPD) within DHFR was identical in tGM24 and GM00024 cells. CPD removal from the transcribed strand was nearly complete in 6h and from the non-transcribed strand was 73% complete in 24h. UV-induced HPRT mutagenesis in tGM24 was indistinguishable from primary human fibroblasts. These wide-ranging findings indicate that the UV-induced DNA damage response remains intact in telomerase-immortalized cells. Furthermore, telomerase immortalization provides permanent cell lines for testing the immediate impact on NER and mutagenesis of selective genetic manipulation without propagation to establish mutant lines.     

Chromatin restoration following nucleotide excision repair involves the incorporation of ubiquitinated H2A at damaged genomic sites

Restoration of functionally intact chromatin structure following DNA damage processing is crucial for maintaining genetic and epigenetic information in human cells. Here, we show the UV-induced uH2A foci formation in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF indicating that uH2A incorporation relied on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for formation of γH2AX foci in asynchronous cells. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone CAF-1 showed distinct localization to the damage sites. Knockdown of CAF-1 p60 abolished CAF-1 as well as uH2A foci formation. CAF-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. These data demonstrate that successful NER of genomic lesions and prompt CAF-1-mediated chromatin restoration link uH2A incorporation at the sites of damage repair within chromatin.     

Ubiquitin-specific Protease 7 Regulates Nucleotide Excision Repair through Deubiquitinating XPC Protein and Preventing XPC Protein from Undergoing Ultraviolet Light-induced and VCP/p97 Protein-regulated Proteolysis

Ubiquitin specific protease 7 (USP7) is a known deubiquitinating enzyme for tumor suppressor p53 and its downstream regulator, E3 ubiquitin ligase Mdm2. Here we report that USP7 regulates nucleotide excision repair (NER) via deubiquitinating xeroderma pigmentosum complementation group C (XPC) protein, a critical damage recognition factor that binds to helix-distorting DNA lesions and initiates NER. XPC is ubiquitinated during the early stage of NER of UV light-induced DNA lesions. We demonstrate that transiently compromising cellular USP7 by siRNA and chemical inhibition leads to accumulation of ubiquitinated forms of XPC, whereas complete USP7 deficiency leads to rapid ubiquitin-mediated XPC degradation upon UV irradiation. We show that USP7 physically interacts with XPC in vitro and in vivo. Overexpression of wild-type USP7, but not its catalytically inactive or interaction-defective mutants, reduces the ubiquitinated forms of XPC. Importantly, USP7 efficiently deubiquitinates XPC-ubiquitin conjugates in deubiquitination assays in vitro. We further show that valosin-containing protein (VCP)/p97 is involved in UV light-induced XPC degradation in USP7-deficient cells. VCP/p97 is readily recruited to DNA damage sites and colocalizes with XPC. Chemical inhibition of the activity of VCP/p97 ATPase causes an increase in ubiquitinated XPC on DNA-damaged chromatin. Moreover, USP7 deficiency severely impairs the repair of cyclobutane pyrimidine dimers and, to a lesser extent, affects the repair of 6-4 photoproducts. Taken together, our findings uncovered an important role of USP7 in regulating NER via deubiquitinating XPC and by preventing its VCP/p97-regulated proteolysis.     

p53-degradation by HPV-16 E6 preferentially affects the removal of cyclobutane pyrimidine dimers from non-transcribed strand and sensitizes mammary epithelial cells to UV-irradiation

Nucleotide excision repair (NER), the most versatile and ubiquitous mechanism for DNA repair, operates to remove many types of DNA base lesions. We have studied the role of p53 function in modulating the repair of DNA damage following UV irradiation in normal and p53-compromised human mammary epithelial cells (HMEC). The effect of UV-induced DNA damage on cellular cytotoxicity and apoptosis was determined in conjunction with global, gene- and strand-specific repair. Cytotoxicity studies, using clonogenic survival and MTT assays, showed that HPV-16 E6-expressing HMEC were more UV sensitive than p53-WT cell lines. High apoptotic index obtained with p53-compromised cells was in conformity to both the low clonogenic survival and the low cellular viability. No discernible differences in the formation of initial UV-induced cyclobutane pyrimidine dimers (CPD) were observed in the cell lines of varying p53 functional status. However, the extent and the rate of damage removal from genome overall were highest for p53-WT cells. Further examination of strand-specific repair in the p53 gene revealed that the removal of CPD in the non-transcribed strand (NTS) was slower in p53-compromised cells compared to the normal p53-WT cell lines. These results suggest that loss of p53 function, in the absence of other genetic alterations, decreased both overall amount of CPD repaired and their removal rate from the genome. Additionally, normal function of p53 is required for the repair of the NTS, but not of the transcribed strand (TS) in genomic DNA in human epithelial cells. Thus, failure of quantitative removal of CPD by global genomic repair (GGR), due to loss of p53 function, causes the enhanced UV sensitivity and increased damage-induced apoptosis via a p53-independent pathway. Nevertheless, recovery of cells from UV damage requires normal p53 function and efficient GGR.     

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.     

Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites

Nucleotide excision repair (NER) of DNA damage requires an efficient means of discrimination between damaged and non-damaged DNA. Cells from humans with xeroderma pigmentosum group C do not perform NER in the bulk of the genome and are corrected by XPC protein, which forms a complex with hHR23B protein. This complex preferentially binds to some types of damaged DNA, but the extent of discrimination in comparison to other NER proteins has not been clear. Recombinant XPC, hHR23B, and XPC-hHR23B complex were purified. In a reconstituted repair system, hHR23B stimulated XPC activity tenfold. Electrophoretic mobility-shift competition measurements revealed a 400-fold preference for binding of XPC-hHR23B to UV damaged over non-damaged DNA. This damage preference is much greater than displayed by the XPA protein. The discrimination power is similar to that determined here in parallel for the XP-E factor UV-DDB, despite the considerably greater molar affinity of UV-DDB for DNA. Binding of XPC-hHR23B to UV damaged DNA was very fast. Damaged DNA-XPC-hHR23B complexes were stable, with half of the complexes remaining four hours after challenge with excess UV-damaged DNA at 30 degrees C. XPC-hHR23B had a higher level of affinity for (6-4) photoproducts than cyclobutane pyrimidine dimers, and some affinity for DNA treated with cisplatin and alkylating agents. XPC-hHR23B could bind to single-stranded M13 DNA, but only poorly to single-stranded homopolymers. The strong preference of XPC complex for structures in damaged duplex DNA indicates its importance as a primary damage recognition factor in non-transcribed DNA during human NER.