Spatial organization of nucleotide excision repair proteins after UV-induced DNA damage in the human cell nucleus (original) (raw)
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Mutation Research/DNA Repair, 1997
We have used the buoyant density shift method to measure excision-repair patch lengths in UV-irradiated repair-profi-Ž . cient human cells and in primary fibroblasts belonging to xeroderma pigmentosum complementation group C XP-C , in which excision repair of UV-induced photoproducts is dependent upon transcription. The patch size was found to be about 30 nucleotides for both cell types. This agrees with the size of the DNA fragments excised in vitro by the dual incisions of the structure-specific nucleases XPG and ERCC1-XPF. We conclude that the XPC protein is not required to target the excision nucleases to sites of DNA cleavage in transcribed strands of expressed genes or to protect the newly incised DNA from further processing by exonucleases. q 1997 Elsevier Science B.V. 0921-8777r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved.
DNA Repair, 2017
Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type.
DNA Repair, 2011
a b s t r a c t DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of ␥-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6 h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24 h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6 h and 24 h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6 h and 24 h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.
Sequential Assembly of the Nucleotide Excision Repair Factors In Vivo
Molecular Cell, 2001
Leiden University Medical Center ema et al., 1992). Examination of the repair kinetics of Wassenaarseweg 72 structurally different DNA lesions has disclosed that 2333 AL, Leiden TCR functions as an efficient repair system for transcrip-2 Swammerdam Institute for Life Sciences tion-blocking lesions that are poorly repaired by GGR BioCentrum Amsterdam (Hanawalt, 1995). In the case of UV-induced photole-University of Amsterdam sions, repair of 6-4PP is fast throughout the genome Plantage Muidergracht 12 and is dominated by GGR. In contrast, GGR of CPD is 1018 TV, Amsterdam relatively slow, but TCR causes accelerated removal of 3 Department of Cell Biology and Genetics this lesion from the transcribed strand of expressed Medical Genetics Center genes (van Hoffen et al., 1995). Erasmus University Rotterdam Cell fusion experiments have revealed seven XP ge-PO Box 1738 netic complementation groups (XP-A through XP-G) that 3000 DR, Rotterdam represent different proteins in the NER pathway. Among The Netherlands the various complementation groups, XP-C is unique, as only GGR is compromised in this group (Venema et al., 1991). The photosensitive inherited disorder Cock-Summary ayne syndrome (CS), on the other hand, is associated with defective TCR, while GGR is unaffected (van Hoffen Here, we describe the assembly of the nucleotide exciet al., 1993; Venema et al., 1990). At the cellular level, sion repair (NER) complex in normal and repair-defithe two CS genetic complementation groups (CS-A and cient (xeroderma pigmentosum) human cells, em-CS-B) are characterized by a lack of recovery of inhibited ploying a novel technique of local UV irradiation RNA synthesis following exposure to DNA damaging combined with fluorescent antibody labeling. The agents, a phenomenon that has been related to defecdamage recognition complex XPC-hHR23B appears tive TCR (Mayne and Lehmann, 1982; Venema et al., to be essential for the recruitment of all subsequent 1990). NER factors in the preincision complex, including tran-Incision of damaged DNA is a multistep process inscription repair factor TFIIH. XPA associates relatively volving recognition of the DNA damage followed by late, is required for anchoring of ERCC1-XPF, and may opening up of the DNA helix around the lesion, dual be essential for activation of the endonuclease activity incision, and subsequent excision of the oligonucleotide of XPG. These findings identify XPC as the earliest containing the DNA lesion (de Laat et al., 1999). From known NER factor in the reaction mechanism, give in vitro biochemistry, it is not clear in which order various insight into the order of subsequent NER components, NER factors act in the reaction mechanism, particularly provide evidence for a dual role of XPA, and support with respect to the first stages including the crucial a concept of sequential assembly of repair proteins damage recognition step. In addition, it is not evident at the site of the damage rather than a preassembled what the organization of repair is in vivo-are NER facrepairosome. tors preassembled in a NER holocomplex, in distinct subassemblies, or as individual factors that transiently Introduction interact at the site of the lesion? The identity of the damage recognition factor has been a matter of debate. In eukaryotes, nucleotide excision repair (NER) is a ver-Several putative candidates have been proposed, insatile and highly conserved repair system capable of cluding the XPA-replication protein A (RPA) complex removing a wide range of DNA lesions that distort the (Asahina et al., 1994; Li et al., 1995b), the XPC-hHR23B stacking of the DNA double helix, including the shortcomplex (Reardon et al., 1996, Batty and Wood, 2000; wave ultraviolet (UV) light-induced cyclobutane pyrimi-Yokoi et al., 2000), and the p48-p127 complex, also dine dimers (CPD) and 6-4 photoproducts (6-4PP). In termed damaged DNA binding (DDB) protein (Chu and humans, repair of UV-induced photolesions is entirely Chang, 1988; Tang et al., 2000). DDB has not been implidependent on NER, and mutations in NER proteins have cated so far in the damage recognition step in in vitro been associated with the inherited disorder xeroderma experiments. Results obtained in in vitro experiments pigmentosum (XP) (Bootsma et al., 1998). XP patients are contradictory as to whether XPC-hHR23B or XPA-RPA is the principal damage recognition protein. Findings by Sugasawa and coworkers using N-acetoxy-2-4 Correspondence: zeeland@lumc.nl
Experimental Cell Research, 2002
The ubiquitous process of nucleotide excision repair includes an obligatory step of DNA repair synthesis (DRS) to fill the gapped heteroduplex following excision of a short (ϳ30-nucleotide) damaged single-strand fragment. Using 5-iododeoxyuridine to label repair patches during the first 10 -60 min after UV irradiation of quiescent normal human fibroblasts we have visualized a limited number of discrete foci of DRS. These must reflect clusters of elementary DRS patches, since single patches would not be detected. The DRS foci are attenuated in normal cells treated with ␣-amanitin or in Cockayne syndrome (CS) cells, which are specifically deficient in the pathway of transcription-coupled repair (TCR). It is therefore likely that the clusters of DRS arise in chromatin domains within which RNA polymerase II transcription is compartmentalized. However, we also found significant suppression of DRS foci in xeroderma pigmentosum, complementation group C cells in which global genome repair (GGR) is defective, but TCR is normal. This suggests that the TCR is responsible for the DRS cluster formation in the absence of GGR. The residual foci detected in CS cells indicate that, even at early times following UV irradiation, GGR may open some chromatin domains for processive scanning and consequent DRS independent of transcription. © 2002 Elsevier Science (USA)
Low amounts of the DNA repair XPA protein are sufficient to recover UV-resistance
Carcinogenesis, 2002
Nucleotide excision repair (NER) is one of the most known and flexible mechanisms of DNA repair. This mechanism can recognize and remove damages causing DNA doublehelix distortion, including the cyclobutane pyrimidine dimers (CPDs) and the pyrimidine-pyrimidone (6-4) photoproducts, promoted by ultraviolet light (UV). The human syndrome xeroderma pigmentosum (XP) is clinically characterized chiefly by the early onset of severe photosensitivity of the exposed regions of the skin, a very high incidence of skin cancers and frequent neurological abnormalities. The xpa gene seems to be involved during UV damage recognition, in both global genome repair (GGR) and transcription-coupled repair (TCR). The modulation of xpa expression may modify the DNA repair rate in the cell genome, providing a valuable contribution to an understanding of the NER process. The controlled expression of the cDNA xpa in XP12RO deficient cells was achieved through the transfection of a muristerone-A inducible vector, pINXA. The INXA15 clone shows good induction of the XPA protein and total complementation of XP12RO cell deficiency. Overexpression of this protein resulted in UV cell survival comparable to normal control human cells. Moreover, low expression of the XPA protein in these cells is sufficient for total complementation in cellular UV sensitivity and DNA repair activity. These data demonstrate that XPA protein concentration is not a limiting factor for DNA repair.
Journal of Biological Chemistry, 2007
In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38␣/ become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38␣/ to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38␣ mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair. Nucleotide excision repair (NER) 3 is the only pathway available to human cells for the removal of helix-distorting (replication-and transcription-blocking) "bulky" DNA adducts generated by a multitude of environmental carcinogens. Among these adducts is the highly promutagenic UV-induced cyclobutane pyrimidine dimer (CPD), which lies at the origin of sunlight-associated mutagenesis and skin cancer development (1). The physiological importance of NER is highlighted by xeroderma pigmentosum (XP), a rare genetic disorder characterized by defective removal of bulky DNA adducts, UV hypersensitivity, and striking predisposition to skin cancer (2). Furthermore, NER status of tumors in cancer patients has been identified as a major determinant in the clinical response to UV-mimetic chemotherapeutic agents, such as cisplatin, which exert antineoplastic effects via the induction of bulky DNA adducts (3). NER is composed of two distinct subpathways (i.e. global genomic-NER (GG-NER) removes bulky adducts from the genome overall, whereas transcription-coupled NER (TC-NER) removes such adducts exclusively from the transcribed strands of active genes) (see Ref. 4 for an excellent overview). These subpathways differ only in the mechanism of lesion recognition. GG-NER is triggered when the UV-DDB1/UV-DDB2 heterodimer recognizes and binds the helical distortion created by bulky adducts, which is followed by recruitment of the XPC-hHR23B complex. On the other hand, TC-NER is initiated uniquely by blockage of RNA polymerase II and subsequent recruitment of the CS-A and CS-B gene products. Thereafter, in the case of either GG-NER or TC-NER, the common "core NER pathway" is recruited to faithfully restore the integrity of the DNA through sequential steps of localized strand unwinding, incision of the DNA on either side of the adduct, excision of the adduct leaving a small single-stranded gap, and, finally, gap filling and ligation using normal DNA replication factors and the intact complementary strand as template.
Oxidative Medicine and Cellular Longevity
The continuous exposure of the human body’s cells to radiation and genotoxic stresses leads to the accumulation of DNA lesions. Fortunately, our body has several effective repair mechanisms, among which is nucleotide excision repair (NER), to counteract these lesions. NER includes both global genome repair (GG-NER) and transcription-coupled repair (TC-NER). Deficiencies in the NER pathway underlie the development of several DNA repair diseases, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). Deficiencies in GG-NER and TC-NER render individuals to become prone to cancer and neurological disorders, respectively. Therefore, NER regulation is of interest in fine-tuning these risks. Distinct signaling cascades including the NFE2L2 (NRF2), AHR, PI3K/AKT1, MAPK, and CSNK2A1 pathways can modulate NER function. In addition, several chemical and biological compounds have proven success in regulating NER’s activity. These modulators, particularly the ...
UV-DDB-dependent regulation of nucleotide excision repair kinetics in living cells
DNA Repair, 2009
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.