Nucleotide Excision Repair Components Research Articles

Open gaps in the evolution of the eukaryotic nucleotide excision repair

Nucleotide excision repair (NER) is the most versatile DNA repair pathway as it removes different kinds of bulky lesions. Due to its essential role for genome integrity, it has appeared early in the evolution of species. However, most published studies are focused on humans, mice, yeast or bacteria. Considering the large amount of information on genome databases, it is currently possible to retrieve sequences from NER components in many organisms. Therefore, we have characterized the potential orthologs of 10 critical components of the human NER pathway in 12 eukaryotic species by using similarity and structural criteria through the use of bioinformatics tools. This approach has allowed us to characterize gene and protein structures comparatively, taking a glance at some evolutionary aspects of the NER pathway. We have obtained significant search results for the majority of the proteins in most of the organisms studied, mainly for factors that play a pivotal role in the pathway. However, we have revisited significant differences and found new aspects that may imply a distinct functioning of this pathway in different organisms. Through the demonstration of the heterogeneity of the gene structures and a variety in the protein architecture of the NER components evaluated, our results show important differences between human NER and evolutionarily distant eukaryotes. We highlight the lack of a canonical XPD in chicken, the divergence of XPA in plants and protozoans and the absence of XPE in the invertebrate species analyzed. In spite of this, it is remarkable the presence of this excision repair mechanism in a high number of evolutionary distant organisms, being present since the origin of eukaryotes.

Expression of nucleotide excision repair in Alzheimer’s disease is higher in brain tissue than in blood

Age-related changes are increased in patients with Alzheimer’s disease (AD), including oxidative stress and DNA damage. We propose that genotoxic stress and DNA repair responses influence neurodegeneration in the pathogenesis of AD. Here, we focus on nucleotide excision repair (NER). Real-time qPCR and mass spectrometry were employed to determine the expression levels of selected NER components. The mRNA levels of the genes encoding the NER proteins RAD23B, RPA1, ERCC1, PCNA and LIG3 as well as the NER-interacting base excision repair protein MPG in blood and brain tissue from four brain regions in patients with AD or mild cognitive impairment and healthy controls (HC), were assessed. NER mRNA levels were significantly higher in brain tissue than in blood. Further, LIG3 mRNA levels in the frontal cortex was higher in AD versus HC, while mRNA levels of MPG and LIG3 in entorhinal cortex and RPA1 in the cerebellum were lower in AD versus HC. In blood, RPA1 and ERCC1 mRNA levels were lower in AD patients than in HC. Alterations in gene expression of NER components between brain regions were associated with AD, connecting DNA repair to AD pathogenesis and suggesting a distinct role for NER in the brain.

Mycobacterium tuberculosis UvrB forms dimers in solution and interacts with UvrA in the absence of ligands.

During its life cycle Mycobacterium tuberculosis (MTB) must face a variety of environmental and endogenous physical and chemical stresses that could produce genotoxic damage. However, MTB possesses efficient systems to counteract the harmful effects of DNA-damaging assaults. The nucleotide excision repair (NER) is a highly conserved multi-enzymatic cascade that is initiated by the concerted action of three core proteins, that is UvrA, UvrB, and UvrC. Although the functional roles of these enzymes are well characterized, the intra-pathway coordination of the NER components and the dynamics of their association is still a matter of debate. In the presented study, we analyzed the hydrodynamic properties and the oligomeric state of the MTB UvrB protein (MtUvrB) that we expressed and purified to homogeneity in a tag-free form. Our results show that, differently to what has been previously observed for the His-tagged version of the protein, MtUvrB forms dimers in solution, which are characterized by an elongated shape, as determined by small-angle X-ray scattering analysis. Moreover, to gain insights into the mycobacterial UvrA/UvrB lesion sensing/tracking complex we adopted a size-exclusion chromatography-based approach, revealing that the two proteins interact in the absence of ligands, leading to the assembling of A2 B2 hetero-tetramers in solution. Surface plasmon resonance analysis showed that the dissociation constant of the MtUvrA/MtUvrB complex falls in the low micromolar range that could represent the basis for a fine modulation of the complex architecture accompanying the multi-step DNA repair activity of mycobacterial NER.

Nucleotide Excision Repair and Vitamin D--Relevance for Skin Cancer Therapy.

Ultraviolet (UV) radiation is involved in almost all skin cancer cases, but on the other hand, it stimulates the production of pre-vitamin D3, whose active metabolite, 1,25-dihydroxyvitamin D3 (1,25VD3), plays important physiological functions on binding with its receptor (vitamin D receptor, VDR). UV-induced DNA damages in the form of cyclobutane pyrimidine dimers or (6-4)-pyrimidine-pyrimidone photoproducts are frequently found in skin cancer and its precursors. Therefore, removing these lesions is essential for the prevention of skin cancer. As UV-induced DNA damages are repaired by nucleotide excision repair (NER), the interaction of 1,25VD3 with NER components can be important for skin cancer transformation. Several studies show that 1,25VD3 protects DNA against damage induced by UV, but the exact mechanism of this protection is not completely clear. 1,25VD3 was also shown to affect cell cycle regulation and apoptosis in several signaling pathways, so it can be considered as a potential modulator of the cellular DNA damage response, which is crucial for mutagenesis and cancer transformation. 1,25VD3 was shown to affect DNA repair and potentially NER through decreasing nitrosylation of DNA repair enzymes by NO overproduction by UV, but other mechanisms of the interaction between 1,25VD3 and NER machinery also are suggested. Therefore, the array of NER gene functioning could be analyzed and an appropriate amount of 1.25VD3 could be recommended to decrease UV-induced DNA damage important for skin cancer transformation.

Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells

Nucleotide excision repair (NER) protects against sunlight-induced skin cancer. Defective NER is associated with photosensitivity and a high skin cancer incidence. Some clinical treatments that cause photosensitivity can also increase skin cancer risk. Among these, the immunosuppressant azathioprine and the fluoroquinolone antibiotics ciprofloxacin and ofloxacin interact with UVA radiation to generate reactive oxygen species that diminish NER capacity by causing protein damage. The replication protein A (RPA) DNA-binding protein has a pivotal role in DNA metabolism and is an essential component of NER. The relationship between protein oxidation and NER inhibition was investigated in cultured human cells expressing different levels of RPA. We show here that RPA is limiting for NER and that oxidative damage to RPA compromises NER capability. Our findings reveal that cellular RPA is surprisingly vulnerable to oxidation, and we identify oxidized forms of RPA that are associated with impaired NER. The vulnerability of NER to inhibition by oxidation provides a connection between cutaneous photosensitivity, protein damage, and increased skin cancer risk. Our findings emphasize that damage to DNA repair proteins, as well as to DNA itself, is likely to be an important contributor to skin cancer risk.

Combination of Aβ Secretion and Oxidative Stress in an Alzheimer-Like Cell Line Leads to the Over-Expression of the Nucleotide Excision Repair Proteins DDB2 and XPC.

Repair of oxidative DNA damage, particularly Base Excision Repair (BER), impairment is often associated with Alzheimer’s disease pathology. Here, we aimed at investigating the complete Nucleotide Excision Repair (NER), a DNA repair pathway involved in the removal of bulky DNA adducts, status in an Alzheimer-like cell line. The level of DNA damage was quantified using mass spectrometry, NER gene expression was assessed by qPCR, and the NER protein activity was analysed through a modified version of the COMET assay. Interestingly, we found that in the presence of the Amyloid β peptide (Aβ), NER factors were upregulated at the mRNA level and that NER capacities were also specifically increased following oxidative stress. Surprisingly, NER capacities were not differentially improved following a typical NER-triggering of ultraviolet C (UVC) stress. Oxidative stress generates a differential and specific DNA damage response in the presence of Aβ. We hypothesized that the release of NER components such as DNA damage binding protein 2 (DDB2) and Xeroderma Pigmentosum complementation group C protein (XPC) following oxidative stress might putatively involve their apoptotic role rather than DNA repair function.

Comparative insight into nucleotide excision repair components of Plasmodium falciparum.

Nucleotide excision repair (NER) is one of the DNA repair pathways crucial for maintenance of genome integrity and deals with repair of DNA damages arising due to exogenous and endogenous factors. The multi-protein transcription initiation factor TFIIH plays a critical role in NER and transcription and is highly conserved throughout evolution. The malaria parasite Plasmodium falciparum has been a challenge for the researchers for a long time because of emergence of drug resistance. The availability of its genome sequence has opened new avenues for research. Antimalarial drugs like chloroquine and mefloquine have been reported to inhibit NER pathway mediated repair reactions and thus promote mutagenesis. Previous studies have validated existence and implied possible association of defective or altered DNA repair pathways with development of drug resistant phenotype in certain P. falciparum strains. We conjecture that a compromised NER pathway in combination with other DNA repair pathways might be conducive for the emergence and sustenance of drug resistance in P. falciparum. Therefore we decided to unravel the components of NER pathway in P. falciparum and using bioinformatics based approaches here we report a genome wide in silico analysis of NER components from P. falciparum and their comparison with the human host. Our results reveal that P. falciparum genome contains almost all the components of NER but we were unable to find clear homologue for p62 and XPC in its genome. The structure modeling of all the components further suggests that their structures are significantly conserved. Furthermore this study lays a foundation to perform similar comparative studies between drug resistant and drug sensitive strains of parasite in order to understand DNA repair-related mechanisms of drug resistance.

Conservation of the nucleotide excision repair pathway: characterization of hydra Xeroderma Pigmentosum group F homolog.

Hydra, one of the earliest metazoans with tissue grade organization and nervous system, is an animal with a remarkable regeneration capacity and shows no signs of organismal aging. We have for the first time identified genes of the nucleotide excision repair (NER) pathway from hydra. Here we report cloning and characterization of hydra homolog of xeroderma pigmentosum group F (XPF) gene that encodes a structure-specific 5′ endonuclease which is a crucial component of NER. In silico analysis shows that hydra XPF amino acid sequence is very similar to its counterparts from other animals, especially vertebrates, and shows all features essential for its function. By in situ hybridization, we show that hydra XPF is expressed prominently in the multipotent stem cell niche in the central region of the body column. Ectoderm of the diploblastic hydra was shown to express higher levels of XPF as compared to the endoderm by semi-quantitative RT-PCR. Semi-quantitative RT-PCR analysis also demonstrated that interstitial cells, a multipotent and rapidly cycling stem cell lineage of hydra, express higher levels of XPF mRNA than other cell types. Our data show that XPF and by extension, the NER pathway is highly conserved during evolution. The prominent expression of an NER gene in interstitial cells may have implications for the lack of senescence in hydra.

Prokaryotic Nucleotide Excision Repair

Nucleotide excision repair (NER) has allowed bacteria to flourish in many different niches around the globe that inflict harsh environmental damage to their genetic material. NER is remarkable because of its diverse substrate repertoire, which differs greatly in chemical composition and structure. Recent advances in structural biology and single-molecule studies have given great insight into the structure and function of NER components. This ensemble of proteins orchestrates faithful removal of toxic DNA lesions through a multistep process. The damaged nucleotide is recognized by dynamic probing of the DNA structure that is then verified and marked for dual incisions followed by excision of the damage and surrounding nucleotides. The opposite DNA strand serves as a template for repair, which is completed after resynthesis and ligation.

Regulation of nucleotide excision repair through ubiquitination

Nucleotide excision repair (NER) is the most versatile DNA-repair pathway in all organisms. While bacteria require only three proteins to complete the incision step of NER, eukaryotes employ about 30 proteins to complete the same step. Here we summarize recent studies demonstrating that ubiquitination, a post-translational modification, plays critical roles in regulating the NER activity either dependent on or independent of ubiquitin-proteolysis. Several NER components have been shown as targets of ubiquitination while others are actively involved in the ubiquitination process. We argue through this analysis that ubiquitination serves to coordinate various steps of NER and meanwhile connect NER with other related pathways to achieve the efficient global DNA-damage response.

Role of Endonucleases XPF and XPG in Nucleotide Excision Repair of Platinated DNA and Cisplatin/Oxaliplatin Cytotoxicity

Resistance of tumor cells to platinum anticancer agents poses a major problem in cancer chemotherapy. One of the mechanisms associated with platinum-based drug resistance is the enhanced capacity of the cell to carry out nucleotide excision repair (NER) on platinum-damaged DNA. Endonucleases XPF and XPG are critical components of NER, responsible for excising the damaged DNA strand to remove the DNA lesion. Here, we investigated possible consequences of down-regulation of XPF and XPG gene expression in osteosarcoma cancer cells (U2OS) and the impact on cellular transcription and DNA repair. We further evaluated the sensitivity of such cells toward the platinum anticancer drugs cisplatin and oxaliplatin.

Nucleotide Excision Repair by Mutant Xeroderma Pigmentosum Group A (XPA) Proteins with Deficiency in Interaction with RPA

The xeroderma pigmentosum group A protein (XPA) is a core component of nucleotide excision repair (NER). To coordinate early stage NER, XPA interacts with various proteins, including replication protein A (RPA), ERCC1, DDB2, and TFIIH, in addition to UV-damaged or chemical carcinogen-damaged DNA. In this study, we investigated the effects of mutations in the RPA binding regions of XPA on XPA function in NER. XPA binds through an N-terminal region to the middle subunit (RPA32) of the RPA heterotrimer and through a central region that overlaps with its damaged DNA binding region to the RPA70 subunit. In cell-free NER assays, an N-terminal deletion mutant of XPA showed loss of binding to RPA32 and reduced DNA repair activity, but it could still bind to UV-damaged DNA and RPA. In contrast, amino acid substitutions in the central region reduced incisions at the damaged site in the cell-free NER assay, and four of these mutants (K141A, T142A, K167A, and K179A) showed reduced binding to RPA70 but normal binding to damaged DNA. Furthermore, mutants that had one of the four aforementioned substitutions and an N-terminal deletion exhibited lower DNA incision activity and binding to RPA than XPA with only one of these substitutions or the deletion. Taken together, these results indicate that XPA interaction with both RPA32 and RPA70 is indispensable for NER reactions.

Structure of UvrA nucleotide excision repair protein in complex with modified DNA

One of the primary pathways for removal of DNA damage is nucleotide excision repair (NER). In bacteria, the UvrA protein is the component of NER that locates the lesion. A notable feature of NER is its ability to act on many DNA modifications that vary in chemical structure. So far, the mechanism underlying this broad specificity has been unclear. Here, we report the first crystal structure of a UvrA protein in complex with a chemically modified oligonucleotide. The structure shows that the UvrA dimer does not contact the site of lesion directly, but rather binds the DNA regions on both sides of the modification. The DNA region harboring the modification is deformed, with the double helix bent and unwound. UvrA uses damage-induced deformations of the DNA and a less rigid structure of the modified double helix for indirect readout of the lesion.

Fission yeast homologs of human XPC and CSB, rhp41 and rhp26, are involved in transcription-coupled repair of methyl methanesulfonate-induced DNA damage

Methyl methanesulfonate (MMS) methylates nitrogen atoms in purines, and predominantly produces 7-methylguanine and 3-methyladenine (3-meA). Previously, we showed that base excision repair (BER) and nucleotide excision repair (NER) synergistically function to repair MMS-induced DNA damage in the fission yeast Schizosaccharomyces pombe. Here, we studied the roles of NER components in repair of 3-meA and BER intermediates such as the AP site and single strand breaks. Mutants of rhp41 (XPC homolog) and rhp26 (CSB homolog) exhibited moderate sensitivity to MMS. Transcription of the fbp1 gene, which is induced by glucose starvation, was strongly inhibited by MMS damage in rhp41Δ and rhp26Δ strains but not in wild type and 3-meA DNA glycosylase-deficient cells. The results indicate that Rhp41p and Rhp26p are involved in transcription-coupled repair (TCR) of MMS-induced DNA damage. In the BER pathway of S. pombe, AP lyase activity of Nth1p mainly incises the AP site to generate a 3'-blocked end, which is in turn converted to 3'-OH by Apn2p. Deletion of rad16 or rhp26 in the nth1Δ strain greatly enhanced MMS sensitivity, suggesting that the AP site could also be corrected by TCR. Double mutant apn2Δ/rad16Δ exhibited hypersensitivity to MMS, implying that Rad16p provides a backup pathway for removal of the 3'-blocked end. Moreover, an rhp51Δ strain was extremely sensitive to MMS and double mutants of nth1Δ/rhp51Δ and apn2Δ/rhp51Δ increased the sensitivity, suggesting that homologous recombination is necessary for repair of three different types of lesions, 3-meA, AP sites and 3'-blocked ends.

Expression of ERCC1 in triple-negative breast cancer.

e11064 Background: Triple-negative breast cancer (TNBC) comprises 15% of breast cancers. Since TNBC lack the available targets of treatment, cytotoxic therapy is the standard approach. Platinum-based chemotherapy regimens seem promising in this setting. Excision repair cross-complementation group 1 (ERCC1) which is a component of nucleotide excision repair (NER) pathway removes platinum induced DNA adducts. Over expression of ERCC1 has been associated with resistance to platinum-based chemotherapy in ovarian and lung cancers. Detecting ERCC1 over expression is important in planning treatment options for TNBC and conducting trials that are investigating specific chemotherapy regimen for TNBC. Methods: Monoclonal antibody against ERCC1 (clone 8F1, Neomarkers) was used for analyzing immunohistochemical expression of ERCC1. Two experienced pathologists blinded to clinical data evaluated ERCC1 expression in 45 TNBC patients' tumor samples. A semi-quantitative H score was calculated by multiplying staining intensity with extent score. Tumors with H-score ≥ 1 classified as ERCC1 positive. Results: Positive ERCC1 expression was found in 73.3% of the tumor samples with an H score ≥ 1 and 26.7% of the tumor samples stained negative with an H score less than 1. 15.5% of the tumor samples stained diffusely and intensively with ERCC1 antibody. Higher ERCC1 expression tended to be correlated with tumor grade (Pearson's correlation coefficient, 0.22; p = 0.14) and family history of cancer (Pearson's correlation coefficient, 0.29; p = 0.053). ERCC1 expression was not correlated with any of the following clinical parameters; age at diagnosis, menopausal status, stage, history of cancer, recurrence in follow-up period. Conclusions: Our results demonstrate that about two thirds of the TNBC showed a relevant expression of ERCC1 that may be predictive of a poor response to platinum-based chemotherapies. Dysfunction of other DNA repair enzymes such as inactivation of BRCA-1 may explain higher sensitivity to cisplatin in TNBC patients. No significant financial relationships to disclose.

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

The effects of UV irradiation on herpes simplex virus type 1 (HSV-1) gene expression and DNA replication were examined in cell lines containing mutations inactivating the XPA gene product required for nucleotide-excision repair, the DNA polymerase eta responsible for translesion synthesis, or the Cockayne syndrome A and B (CSA and CSB) gene products required for transcription-coupled nucleotide excision repair. In the absence of XPA and CSA and CSB gene products, virus replication was reduced 10(6)-, 400-, and 100-fold, respectively. In DNA polymerase eta mutant cells HSV-1 plaque efficiency was reduced 10(4)-fold. Furthermore, DNA polymerase eta was strictly required for virus replication at low multiplicities of infection but dispensable at high multiplicities of infection. Knock down of Rad 51, Rad 52, and Rad 54 levels by RNA interference reduced replication of UV-irradiated HSV-1 150-, 100-, and 50-fold, respectively. We find that transcription-coupled repair efficiently supports expression of immediate early and early genes from UV-irradiated HSV-1 DNA. In contrast, the progression of the replication fork appears to be impaired, causing a severe reduction of late gene expression. Since the HSV-1 replisome does not make use of proliferating cell nuclear antigen, we attribute the replication defect to an inability to perform proliferating cell nuclear antigen-dependent translesion synthesis by polymerase switching at the fork. Instead, DNA polymerase eta may act during postreplication gap filling. Homologous recombination, finally, might restore the physical and genetic integrity of the virus chromosome.

Preincision Processing of Cyclobutane Pyrimidine Dimers in Escherichia coli

AbstractIn both prokaryotes and eukaryotes cyclobutane pyrimidine dimers (CPD) formed by UV-irradiation are repaired mainly by the nucleotide excision repair (NER) pathway. Evidence has been presented that CPDs are repaired more rapidly and efficiently in vivo than when using purified NER components in vitro, and raises the possibility that CPD can be processed at the preincision stage before being removed by NER pathway. We investigated this hypothesis in E. coli cells and found that the phosphodiester bonds in CPDs are indeed incised (for the sake of simplicity we term this result as CPD*) in a time-dependent fashion in NER-proficient cells. This CPD preincision processing also occurs in NER-deficient uvrB- and uvrC-, but not in uvrA-mutant cells, and in endonuclease V (nfi) mutant cells. Introduction of the nfi mutation into wild type or uvrB-mutant cells results in disablement of CPD to CPD* conversion and enhances UV sensitization to the level of uvrA-mutant cells. We found that in a reconstituted in vitro system, that purified UvrA and Nfi are necessary but not sufficient to convert CPD to CPD*. A UvrC homologous protein, Cho, is also needed for this conversion. In addition, we found that Nfi does not bind to UV-irradiated DNA, suggesting that UvrA and Cho proteins may function as matchmakers for Nfi-CPD binding, which consequently results in the conversion of CPD to CPD*. We further demonstrated that CPD*, but not CPD, can allow efficient translesion bypass synthesis with accuracy. We thus have elucidated a new CPD preincision processing mechanism and genes involved in this process. The biological significance of these findings is discussed elsewhere (another manuscript in preparation).

The molecular basis for different disease states caused by mutations in TFIIH and XPG

Alterations in genes involved in nucleotide excision repair (NER) are associated with three genetic disorders, xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The transcription and repair factor TFIIH is a central component of NER and mutations of its subunits are associated with all three diseases. A recent report provides a molecular basis for how mutations in the NER endonuclease XPG that affect the interaction of TFIIH might give rise to CS features. In cells of XP-G patients with a combined XP and CS phenotype, XPG fails to associate with TFIIH and as a consequence the CAK subunit dissociates from core TFIIH. A simplified, but general model of how various assembly and disassembly states of TFIIH can be invoked to explain different disease states is discussed. Accordingly, defects in specific enzymatic functions typically result in XP, dissociation of the CAK subunit from TFIIH is associated with XP/CS and a more generalized destabilization of TFIIH gives rise to TTD. While this classification provides a useful framework to understand how alterations in TFIIH correlate with disease states, it does not universally apply and relevant exception and alternative explanations are discussed.

Adaptive Response to Gamma Radiation in Mammalian Cells Proficient and Deficient in Components of Nucleotide Excision Repair

Cells preconditioned with low doses of low-linear energy transfer (LET) ionizing radiation become more resistant to later challenges of radiation. The mechanism(s) by which cells adaptively respond to radiation remains unclear, although it has been suggested that DNA repair induced by low doses of radiation increases cellular radioresistance. Recent gene expression profiles have consistently indicated that proteins involved in the nucleotide excision repair pathway are up-regulated after exposure to ionizing radiation. Here we test the role of the nucleotide excision repair pathway for adaptive response to gamma radiation in vitro. Wild-type CHO cells exhibited both greater survival and fewer HPRT mutations when preconditioned with a low dose of gamma rays before exposure to a later challenging dose. Cells mutated for ERCC1, ERCC3, ERCC4 or ERCC5 did not express either adaptive response to radiation; cells mutated for ERCC2 expressed a survival adaptive response but no mutation adaptive response. These results suggest that some components of the nucleotide excision repair pathway are required for phenotypic low-dose induction of resistance to gamma radiation in mammalian cells.

The protein shuffle

Xeroderma pigmentosum (XP) is an inherited disease in which cells from patients exhibit defects in nucleotide excision repair (NER). XP proteins A-G are crucial in the processes of DNA damage recognition and incision, and patients with XP can carry mutations in any of the genes that specify these proteins. In mammalian cells, NER is a dynamic process in which a variety of proteins interact with one another, via modular domains, to carry out their functions. XP proteins are key players in several steps of the NER process, including DNA strand discrimination (XPA, in complex with replication protein A), repair complex formation (XPC, in complex with hHR23B; XPF, in complex with ERCC1) and repair factor recruitment (transcription factor IIH, in complex with XPG). Through these protein-protein interactions, various types of bulky DNA adducts can be recognized and repaired. Communication between the NER system and other cellular pathways is also achieved by selected binding of the various structural domains. Here, we summarize recent studies on the domain structures of human NER components and the regulatory networks that utilize these proteins. Data provided by these studies have helped to illuminate the complex molecular interactions among NER factors in the context of DNA repair.