Regulation Of Nucleotide Excision Repair Research Articles

Epigenetic Regulation of Nucleotide Excision Repair.

Genomic DNA is constantly attacked by a plethora of DNA damaging agents both from endogenous and exogenous sources. Nucleotide excision repair (NER) is the most versatile repair pathway that recognizes and removes a wide range of bulky and/or helix-distorting DNA lesions. Even though the molecular mechanism of NER is well studied through in vitro system, the NER process inside the cell is more complicated because the genomic DNA in eukaryotes is tightly packaged into chromosomes and compacted into a nucleus. Epigenetic modifications regulate gene activity and expression without changing the DNA sequence. The dynamics of epigenetic regulation play a crucial role during the in vivo NER process. In this review, we summarize recent advances in our understanding of the epigenetic regulation of NER.

UV Radiation-induced Impairment of Cellular Morphology and Motility is Enhanced by DUSP3/VHR Loss and FAK Activation.

DUSP3 is a phosphatase expressed and active in several tissues that dephosphorylates tyrosine residues in many regulatory proteins of cellular activities such as proliferation, survival, and cell death. Recently, two new independent functions were assigned to this enzyme: dephosphorylation of focal adhesion kinase (FAK) and regulation of nucleotide-excision repair (NER) pathway. Genotoxic stress by UV radiation is known to affect cell morphology, adhesion, and migration for affecting, for example, the Rho GTPases that regulate actin cytoskeleton. This work investigated the intersection of DUSP3 function, XPA protein activity, and UV toxicity by examining cell migration, FAK, and SRC kinase phosphorylation status, in addition to cell morphology, in fibroblast cells proficient (MRC-5) or deficient (XPA) of the NER pathway. DUSP3 loss reduced cell migration of normal cells, which was stimulated by the genotoxic stress, effects evidenced in presence of serum mitogenic stimulus. However, NER-deficient cells migration response was the opposite since DUSP3 loss increased migration, especially after cells being exposed to UV stress. The levels of pFAK(Y397) peaked 15 min and 1 h after UV radiation in normal cells, but only slightly increased in repair-deficient cells. However, the DUSP3 knockdown strongly raised pFAK(Y397) levels in both cells, but especially in XPA cells as supported by the higher SRC activity. These effects impacted on the dynamics of actin-based structures formation, such as stress fibres, apparently dependent on DUSP3 and DNA-repair (NER) proficiency of the cells. Altogether our findings suggest this dual-phosphatase is bridging gaps between the complex regulation of cell morphology, motility, and genomic stability.

Clinical Perspectives of ERCC1 in Bladder Cancer.

ERCC1 is a key regulator of nucleotide excision repair (NER) pathway that repairs bulky DNA adducts, including intrastrand DNA adducts and interstrand crosslinks (ICLs). Overexpression of ERCC1 has been linked to increased DNA repair capacity and platinum resistance in solid tumors. Multiple single nucleotide polymorphisms (SNPs) have been detected in ERCC1 gene that may affect ERCC1 protein expression. Platinum-based treatment remains the cornerstone of urothelial cancer treatment. Given the expanding application of neoadjuvant and adjuvant chemotherapy in locally advanced bladder cancer, there is an emerging need for biomarkers that could distinguish potential responders to cisplatin treatment. Extensive research has been done regarding the prognostic and predictive role of ERCC1 gene expression and polymorphisms in bladder cancer. Moreover, novel compounds have been recently developed to target ERCC1 protein function in order to maximize sensitivity to cisplatin. We aim to review all the existing literature regarding the role of the ERCC1 gene in bladder cancer and address future perspectives for its clinical application.

Signaling Pathways, Chemical and Biological Modulators of Nucleotide Excision Repair: The Faithful Shield against UV Genotoxicity.

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 positive ones, could therefore provide potential treatments for genetic DNA repair-based diseases. Negative modulators, nonetheless, can help sensitize cells to killing by genotoxic chemicals. In this review, we will summarize and discuss the major upstream signaling pathways and molecules that could modulate the NER's activity.

Abstract 2575: EZH2 mediates resistance to cisplatin in small cell lung cancer through nucleotide excision repair

Abstract Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, with a five-year survival rate of 7%. Cisplatin-based chemotherapy is the first line treatment for SCLC; however, many patients develop treatment resistance and experience tumor recurrence. Targeting proteins critical to the repair of cisplatin DNA crosslinks is a strategy for overcoming acquired cisplatin resistance in SCLC, but many proteins that mediate crosslink repair have yet to be identified. To address this issue, we performed a synthetic lethal siRNA screen in cisplatin resistant SCLC cells, and identified EZH2 as one of the strongest mediators of cisplatin resistance. EZH2 localizes to sites of DNA damage which are induced by UVA-crosslinking laser microirradiation and interacts in a complex with DDB1, and DDB2, members of the nucleotide excision repair (NER) pathway. Loss of EZH2 sensitizes SCLC cells to UV damage, and further, loss of EZH2 and DDB1 together are epistatic in the sensitization of SCLC to cisplatin, confirming a role for EZH2 in NER. Specifically, EZH2 promotes the stability of DDB2, which is responsible for the detection NER lesions. This occurs independently of EZH2’s methyltransferase activity on H3K27. Finally, we found EZH2 expression correlates with cisplatin resistance across SCLC cell lines. Together, this data suggests that EZH2 functions as a novel regulator of NER, and that EZH2 is a promising target for cisplatin resistant SCLC. Citation Format: Allyson E. Koyen. EZH2 mediates resistance to cisplatin in small cell lung cancer through nucleotide excision repair [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2575.

Transcriptional and Posttranslational Regulation of Nucleotide Excision Repair: The Guardian of the Genome against Ultraviolet Radiation.

Ultraviolet (UV) radiation from sunlight represents a constant threat to genome stability by generating modified DNA bases such as cyclobutane pyrimidine dimers (CPD) and pyrimidine-pyrimidone (6-4) photoproducts (6-4PP). If unrepaired, these lesions can have deleterious effects, including skin cancer. Mammalian cells are able to neutralize UV-induced photolesions through nucleotide excision repair (NER). The NER pathway has multiple components including seven xeroderma pigmentosum (XP) proteins (XPA to XPG) and numerous auxiliary factors, including ataxia telangiectasia and Rad3-related (ATR) protein kinase and RCC1 like domain (RLD) and homologous to the E6-AP carboxyl terminus (HECT) domain containing E3 ubiquitin protein ligase 2 (HERC2). In this review we highlight recent data on the transcriptional and posttranslational regulation of NER activity.

Deficient Nucleotide Excision Repair in Squamous Cell Carcinoma Cells.

Squamous cell carcinomas (SCCs) are associated with ultraviolet radiation and multiple genetic changes, but the mechanisms leading to genetic instability are unclear. SCC cell lines were compared to normal keratinocytes for sensitivity to ultraviolet radiation, DNA repair kinetics and DNA repair protein expression. Relative to normal keratinocytes, four SCC cell lines were all variably sensitive to ultraviolet radiation and, except for the SCC25 cell line, were deficient in global repair of cyclobutane pyrimidine dimers, although not 6-4 photoproducts. Impaired DNA repair of cyclobutane pyrimidine dimers was associated with reduced mRNA expression from XPC but not DDB2 genes which each encode key DNA damage recognition proteins. However, levels of XPC or DDB2 proteins or both were variably reduced in repair-deficient SCC cell lines. p53 levels did not correlate with DNA repair activity or with XPC and DDB2 levels, but p63 levels were deficient in cell lines with reduced global repair. Repair-proficient SCC25 cells depleted of p63 lost XPC expression, early global DNA repair activity and UV resistance. These results demonstrate that some SCC cell lines are deficient in global nucleotide excision repair and support a role for p63 as a regulator of nucleotide excision repair in SCCs.

Molecular regulation of UV-induced DNA repair.

Ultraviolet (UV) radiation from sunlight is a major etiologic factor for skin cancer, the most prevalent cancer in the United States, as well as premature skin aging. In particular, UVB radiation causes formation of specific DNA damage photoproducts between pyrimidine bases. These DNA damage photoproducts are repaired by a process called nucleotide excision repair, also known as UV-induced DNA repair. When left unrepaired, UVB-induced DNA damage leads to accumulation of mutations, predisposing people to carcinogenesis as well as to premature aging. Genetic loss of nucleotide excision repair leads to severe disorders, namely, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS), which are associated with predisposition to skin carcinogenesis at a young age as well as developmental and neurological conditions. Regulation of nucleotide excision repair is an attractive avenue to preventing or reversing these detrimental consequences of impaired nucleotide excision repair. Here, we review recent studies on molecular mechanisms regulating nucleotide excision repair by extracellular cues and intracellular signaling pathways, with a special focus on the molecular regulation of individual repair factors.

Gli1 contributes to cellular resistance to cisplatin through altered cellular accumulation of the drug.

Cellular resistance to platinum anticancer compounds is governed by no less than two molecular processes; DNA repair and cellular accumulation of drug. Gli1 is an upstream regulator of nucleotide excision repair, effecting this process through c-jun. We, therefore, investigated whether Gli1 plays a role in cellular accumulation of cisplatin. Using a Gli1-specific shRNA, we explored the role of Gli1 in the cellular accumulation and efflux of cisplatin, in cisplatin-resistant A2780-CP70 human ovarian cancer cells. When Gli1 is inhibited, cellular uptake of cisplatin was approximately 33% of the level of uptake under control conditions. When Gli1 is inhibited, cellular efflux of cisplatin was completely abrogated, over a 12-h period of observation. We assayed nuclear lysates from these cells, for the ability to bind the DNA sequence that is the Gli-binding site (GBS) in the 5′UTR for each of five known cisplatin transmembrane transporters. Four of these transporters are active in cisplatin uptake; and, one is active in cisplatin efflux. In each case, nuclear lysate from A2780-CP70 cells binds the GBS of the respective cisplatin transport gene. We conclude that Gli1 plays a strong role in total cellular accumulation of cisplatin in these cells; and, that the combined effects on cellular accumulation of drug and on DNA repair may indicate a role for Gli1 in protecting cellular DNA from lethal types of DNA damage.

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.

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.

Regulation of Nucleotide Excision Repair by UV-DDB: Prioritization of Damage Recognition to Internucleosomal DNA

Author SummaryLike all molecules in living organisms, DNA undergoes spontaneous decay and is constantly under attack by endogenous and environmental agents. Unlike other molecules, however, DNA—the blueprint of heredity—cannot be re-created de novo; it can only be copied. The original blueprint must therefore remain pristine. All kinds of DNA damage pose a health hazard. DNA lesions induced by the ultraviolet (UV) component of sunlight, for example, can lead to skin aging and skin cancer. A repair process known as nucleotide excision repair (NER) is dedicated to correcting this UV damage. Although the enzymatic steps of this repair process are known in detail, we still do not understand how it copes with the native situation in the cell, where the DNA is tightly wrapped around protein spools called nucleosomes. Our study has revealed the molecular mechanism by which an enigmatic component of NER called UV-DDB stimulates excision of UV-induced lesions in the landscape of nucleosome-packaged DNA in human skin cells. In particular, we describe how this accessory protein prioritizes, in space and time, which UV lesions in packaged DNA to target for repair by NER complexes, thus optimizing the repair process.

GATA-1 Is Essential in EGF-Mediated Induction of Nucleotide Excision Repair Activity and ERCC1 Expression through ERK2 in Human Hepatoma Cells

The nucleotide excision repair (NER) pathway and its leading gene excision-repair cross-complementary 1 (ERCC1) have been shown to be up-regulated in hepatocellular carcinomas even in the absence of treatment with chemotherapeutics. The aim of this study was to determine the mechanism involved in NER regulation during the liver cell growth observed in hepatocellular carcinoma. Both NER activity and ERCC1 expression were increased after exposure to the epidermal growth factor (EGF) in cultured normal and tumoral human hepatocytes. These increases correlated with the activation of the kinase signaling pathway mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK that is known to be a key regulator in the G(1) phase of the hepatocyte cell cycle. Moreover, EGF-mediated activation of ERCC1 was specifically inhibited by either the addition of U0126, a MEK/ERK inhibitor or small interfering RNA-mediated knockdown of ERK2. Basal expression of ERCC1 was decreased in the presence of the phosphoinositide-3-kinase (PI3K) inhibitor and small hairpin RNA (shRNA) against the PI3K pathway kinase FKBP12-rapamycin-associated protein or mammalian target of rapamycin. Transient transfection of human hepatocytes with constructs containing different sizes of the 5'-flanking region of the ERCC1 gene upstream of the luciferase reporter gene showed an increase in luciferase activity in EGF-treated cells, which correlated with the presence of the nuclear transcription factor GATA-1 recognition sequence. The recruitment of GATA-1 was confirmed by chromatin immunoprecipitation assay. In conclusion, these results represent the first demonstration of an up-regulation of NER and ERCC1 in EGF-stimulated proliferating hepatocytes. The transcription factor GATA-1 plays an essential role in the induction of ERCC1 through the mitogen-activated protein kinase (MAPK) pathway, whereas the PI3K signaling pathway contributes to ERCC1 basal expression.

The role of glutathione in the regulation of nucleotide excision repair during oxidative stress

Nucleotide excision repair (NER) mainly repairs bulky DNA adducts and helix distorting lesions, but is additionally considered to be a back-up system for base excision repair to remove oxidative stress induced DNA damage. Therefore, it can be speculated that NER is up-regulated or primed by oxidative stress. Exposure of human pulmonary epithelial cells (A549) to non-toxic doses of 100 μM H 2O 2 indeed showed a 2 to 4.5-fold increase in expression of XPA, XPC, ERCC4, and ERCC5, whereas the expression of ERCC1 was 5-fold decreased. Phenotypical assessment of NER capacity (i.e. recognition and incision of benzo[a]pyrene-DNA adducts) showed a significant decrease to less than 50% after H 2O 2 exposure, which paralleled the effects of H 2O 2 on ERCC1 expression. To study the possible involvement of glutathione (GSH) in the regulation of NER, cells were pre-incubated with 0.5 mM BSO, resulting in total GSH depletion and increased intracellular oxidative stress. In GSH-depleted cells, the down-regulation of ERCC1 expression by H 2O 2 was completely abolished and the up-regulation of ERCC4 expression was potentiated from 2.5-fold to >10-fold. Similarly, the H 2O 2-induced decrease in NER capacity was absent in GSH-depleted cells. Overall, our data suggest that NER capacity as well as the expression of NER related genes can be modulated by oxidative stress. ERCC1 expression and NER capacity correlated strongly ( R 2 = 0.85, P < 0.01) after oxidant exposure, indicating ERCC1 as a specific target for oxidative stress induced modification of NER.

The UBA2 Domain Functions as an Intrinsic Stabilization Signal that Protects Rad23 from Proteasomal Degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

Cellular ubiquitination and proteasomal functions positively modulate mammalian nucleotide excision repair

The ubiquitin-proteasome pathway is fundamental to synchronized continuation of many cellular processes, for example, cell-cycle progression, stress response, and cell differentiation. Recent studies have shown that the ubiquitin-proteasome pathway functions in the regulation of nucleotide excision repair (NER) in yeast. In order to investigate the role of the ubiquitin-proteasome pathway in the NER of mammalian cells, global genomic repair (GGR), and transcription-coupled repair (TCR) were examined in a mouse ts20 cell line that harbors a temperature-sensitive ubiquitin-activating enzyme (E1). We found that E1 inactivation-induced ubiquitination deficiency decreased both GGR and TCR, indicating that the ubiquitination system is involved in the optimization of entire NER machinery in mammalian cells. We specifically inhibited the function of 19S proteasome subunit by overexpressing 19S regulatory complex hSug1 or its mutant protein hSug1mk in repair competent human fibroblast, OSU-2, cells and compared their capacity for NER. The results showed that 19S regulatory complex positively modulates NER in cells. In addition, we treated OSU-2 cells with the inhibitors of 20S subunit function, MG132 and lactacystin, and demonstrated that the catalytic activity of 20S subunit is also required for efficient NER. Moreover, the UV-induced recruitment of repair factor xeroderma pigmentosum protein C (XPC) to damage sites was negatively affected by treatment of repair competent cells with MG132. Taken together, we conclude that the ubiquitin-proteasome pathway has a positive regulatory role for optimal NER capacity in mammalian cells and appears to act through facilitating the recruitment of repair factors to DNA damage sites.

P53 regulation of DNA excision repair pathways.

The regulation of DNA excision repair pathways by p53 and its downstream genes is an emerging body of literature, largely distinct and separable from the more-studied cell cycle arrest and apoptosis responses regulated by p53. Regulation of nucleotide excision repair of UV-damage by p53 and its downstream genes Gadd45 and p48XPE has been well-documented, but much remains to be done in elucidating mechanisms. Moreover, p53 also participates in base excision repair of hydrogen peroxide-induced damage, still at an early stage of investigation. In human cancers carrying inactivating mutations in p53, especially those wherein p53 mutation occurs early, accelerated mutagenesis by exogenous and endogenous DNA damage is predicted. At the same time, the excision repair pathways could provide a useful target for DNA-damaging chemotherapeutics against p53-defective cancers, having decreased ability to repair chemotherapeutic damage. To our knowledge, this is the first review to address this emerging field.

Regulation of nucleotide excision repair in bacteria and mammalian cells.

Regulation of nucleotide excision repair in bacteria and mammalian cells.

P53-dependent regulation of nucleotide excision repair in murine epidermis in vivo.

p53 protects the integrity of the genome by inducing programed cell death or by promoting DNA repair. We have previously shown that loss or mutation of p53 leads to reduced DNA repair in keratinocytes. The hypothesis that p53 regulates repair of ultraviolet light-induced epidermal DNA damage in vivo was tested in mice. An immunohistochemical assay for pyrimidine dimers and 6-4 photoproducts was performed on ultraviolet-irradiated skin from p53 null (-/-) and wild type (+/+) mice. Immunostaining for photoproducts was quantified using computer-assisted imaging. The level of DNA repair was then expressed as the percentage of positive cells remaining as compared to the zero hour time point. p53+/+ mouse skin exposed to 1000 J/m2 retained ' 25% of epidermal cyclobutane dimers at 48 h, whereas approximately 50% remained in p53-/- cells. Using the same UV dose, p53+/+ mice retained 20% of detectable 6-4 photoproducts by 24 h, whereas about 50% remained in epidermal cells of p53-deficient mice. Using in situ labelling of UV-damaged cells, we confirm our earlier conclusion that p53 regulates DNA repair within the epidermis after exposure to UV light.

New patterns of bulk DNA repair in ultraviolet irradiated mouse embryo carcinoma cells following differentiation

Mouse embryocarcinoma stem cells differentiate in culture, given the appropriate induction. We examined whether these cells could provide information about the regulation of nucleotide excision repair in relation to differentiation by measuring the rate-limiting incision step, the removal of cyclobutane dimers and (6-4) photoproducts from the genome as a whole and the effect of the bacteriophage T4 endonuclease (denV) gene on repair in differentiated cells. It was found that differentiation is accompanied by a marked decline in the early incision ability after UV irradiation (sixfold for P19, fourfold for PCC7 and twofold for F9), and we measured, in parallel, the loss of two common UV photoproducts [cyclobutane dimers and (6-4) photoproducts] from P19 cells. After differentiation, the excellent overall cyclobutane dimer repair capacity of proliferating cells (84% removal in 24 h) is lost (no removal in 24 h), while removal of (6-4) photoproducts, although normal at 24 h (94%), is much slower than in undifferentiated P19 at 3 h (no removal versus 64%). The presence of the denV gene greatly stimulates the repair of cyclobutane dimers in undifferentiated P19 cells (94% removal at 3 h versus 40%) and also in differentiated cells (50% removal at 24 h versus no removal). The denV gene also stimulates the early repair of (6-4) photoproducts in both differentiated and undifferentiated cells.