Nucleotide Excision Repair Efficiency Research Articles

Abstract 2034: The NAE1 inhibitor MLN4924 sensitizes both BRCA1 wild type and mutant triple negative breast cancers to cisplatin by reducing the nucleotide excision repair protein XPC level

Abstract Background: Triple-negative breast cancer (TNBC) shows poor survival and prognosis, high recurrence, and poor response to conventional therapeutics. To develop novel therapeutics for TNBC, we explored the efficacy of the neddylation inhibitor, MLN4924. We showed that MLN4924 displayed increased sensitivity against TNBC compared to non-TNBC subtypes. Mechanistically, MLN4924 induced extensive re-replication and DNA damage leading to cell death. Furthermore, MLN4924 improved cisplatin efficacy by elevating the DNA damage levels. To demonstrate the mechanisms of MLN4924+cisplatin-mediated enhanced cell death, we examined the XPC level, a key factor in the nucleotide excision repair (NER) pathway, playing a central role in cisplatin damage repair. Neddylation of CUL4A promotes XPC ubiquitination which influences XPC binding to the DNA damage sites for NER. Methods: We examined the XPC protein level by Western blot assay and RNA level by Real Time PCR using both BRCA1-wild type (MDA-MB-231) and BRCA1-mutated (MDA-MB-436) cell lines and mouse xenograft tumor samples. We evaluated if the proteosome inhibitor MG132 stabilizes XPC ubiquitination and protein levels by treating these cells with MG132 for various times. We also overexpressed XPC in these cell lines and examined the effect on survival upon MLN4924 and MLN4924+cisplatin treatments. Results: Our results demonstrate that MLN4924 reduces XPC ubiquitination upon shorter drug treatment but reduces the protein level upon longer drug treatment in both BRCA1-wild type and BRCA1-mutated cell lines. MG132 treatment did not stabilize the XPC ubiquitination level upon shorter drug treatment but stabilized the protein level upon longer drug treatment. We also observed a reduction in the XPC protein level in tumor samples which correlated with the increase in the DNA damage level. Surprisingly, XPC overexpression improved the cell survival in both MLN4924- and MLN4924+cisplatin-treated BRCA1-wild type and mutated TNBC cells. Conclusion: We hypothesize that a reduction in the XPC ubiquitination and protein levels plays a role in cisplatin-induced DNA damage repair reducing NER efficiency and consequently increasing the DNA damage upon MLN4924+cisplatin treatment. Since overexpression of XPC results in improved survival of both MLN4924-and MLN4924+cisplatin-treated cells, we anticipate that XPC not only regulates cisplatin damage repair through NER but may have a broader role in MLN4924 sensitization beyond the NER pathway. Importantly, MLN4924 treatment does not affect the XPC mRNA level emphasizing that neddylation influences the XPC protein level. In summary, our research uncovers novel insights into the role of XPC in both MLN4924 and MLN4924+cisplatin sensitization, shedding light on potential avenues for innovative therapeutic strategies for TNBC patients. Citation Format: Shrilekha Misra, Mateusz Opyrchal, Alo Ray. The NAE1 inhibitor MLN4924 sensitizes both BRCA1 wild type and mutant triple negative breast cancers to cisplatin by reducing the nucleotide excision repair protein XPC level [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2034.

Noise Stress Abrogates Structure-Specific Endonucleases within the Mammalian Inner Ear.

Nucleotide excision repair (NER) is a multistep biochemical process that maintains the integrity of the genome. Unlike other mechanisms that maintain genomic integrity, NER is distinguished by two irreversible nucleolytic events that are executed by the xeroderma pigmentosum group G (XPG) and xeroderma pigmentosum group F (XPF) structure-specific endonucleases. Beyond nucleolysis, XPG and XPF regulate the overall efficiency of NER through various protein-protein interactions. The current experiments evaluated whether an environmental stressor could negatively affect the expression of Xpg (Ercc5: excision repair cross-complementing 5) or Xpf (Ercc4: excision repair cross-complementing 4) in the mammalian cochlea. Ubiquitous background noise was used as an environmental stressor. Gene expression levels for Xpg and Xpf were quantified from the cochlear neurosensory epithelium after noise exposure. Further, nonlinear cochlear signal processing was investigated as a functional consequence of changes in endonuclease expression levels. Exposure to stressful background noise abrogated the expression of both Xpg and Xpf, and these effects were associated with pathological nonlinear signal processing from receptor cells within the mammalian inner ear. Given that exposure to environmental sounds (noise, music, etc.) is ubiquitous in daily life, sound-induced limitations to structure-specific endonucleases might represent an overlooked genomic threat.

Methods for Assessment of Nucleotide Excision Repair Efficiency.

Nucleotide excision repair (NER) is responsible for removing a wide variety of bulky adducts from DNA, thus contributing to the maintenance of genome stability. The efficiency with which proteins of the NER system recognize and remove bulky adducts depends on many factors and is of great clinical and diagnostic significance. The review examines current concepts of the NER system molecular basis in eukaryotic cells and analyzes methods for the assessment of the NER-mediated DNA repair efficiency both in vitro and ex vivo.

Association between DNA repair capacity and body mass index in women

ObjectiveExamine whether DNA repair capacity (DRC) levels are associated with body mass index (BMI) in adult women. Design and participantsA nested study composed of 539 women without breast cancer (BC) from a case-control BC study in addition to 104 that were recruited later for a total of 643. MeasurementsDRC levels were measured in lymphocytes using a host-cell reactivation assay with a luciferase reporter gene damaged by UVC. This assay measures the efficiency of nucleotide excision repair (NER). Log-binomial regression model was used. The prevalence ratio (PR) was used to evaluate the magnitude of the association between the BMI and DRC levels. An assessment of interaction terms was performed with the likelihood ratio test. The confounding effect was assessed by comparing the point estimates of the crude and adjusted PR. ResultsThe 75th percentiles of DRC levels of the women with a BMI between 18 and 25 and > 25 showed statistically significant differences. The prevalence of a DRC ≤ 5 % among women with BMI > 25 is 1.24 (95 % CI: 1.03, 1.48) times the prevalence of having a DRC ≤ 5 % among the women with BMI ≤ 25 after adjustments for different covariates. This excess was statistically significant (p < 0.05). Women with a family history of cancer had an estimated PR of 1.25 (95 % CI, 0.87–1.39; P ≥ 0.05); and women with no family history of cancer, the estimated PR was 1.6 (95 % CI, 1.14–2.22; p ≤ 0.05). ConclusionsWomen with BMI > 25 tend to have lower DRC levels. When having a family history of cancer, the PR of low DRC levels in overweight/obese individuals was not statistically significant. However, the PR of low levels of DRC in overweight/obese individuals with no family history of cancer was statistically significant.

The Combination of Panobinostat and Melphalan for the Treatment of Patients with Multiple Myeloma.

Histone deacetylase inhibitors show synergy with several genotoxic drugs. Herein, we investigated the biological impact of the combined treatment of panobinostat and melphalan in multiple myeloma (MM). DNA damage response (DDR) parameters and the expression of DDR-associated genes were analyzed in bone marrow plasma cells (BMPCs) and peripheral blood mononuclear cells (PBMCs) from 26 newly diagnosed MM patients. PBMCs from 25 healthy controls (HC) were examined in parallel. Compared with the ex vivo melphalan-only treatment, combined treatment with panobinostat and melphalan significantly reduced the efficiency of nucleotide excision repair (NER) and double-strand-break repair (DSB/R), enhanced the accumulation of DNA lesions (monoadducts and DSBs), and increased the apoptosis rate only in patients' BMPCs (all p &lt; 0.001); marginal changes were observed in PBMCs from the same patients or HC. Accordingly, panobinostat pre-treatment decreased the expression levels of critical NER (DDB2, XPC) and DSB/R (MRE11A, PRKDC/DNAPKc, RAD50, XRCC6/Ku70) genes only in patients' BMPCs; no significant changes were observed in PBMCs from patients or HC. Together, our findings demonstrate that panobinostat significantly increased the melphalan sensitivity of malignant BMPCs without increasing the melphalan sensitivity of PBMCs from the same patients, thus paving the way for combination therapies in MM with improved anti-myeloma efficacy and lower side effects.

Abstract 3747: RPS3 positively regulates nucleotide excision repair with respect to XP-D and skin cancers

Abstract Ribosomal protein S3 (rpS3) is a multifunctional protein working in translation, DNA repair, cancer, apoptosis etc. UV induced DNA damage is repaired by the nucleotide excision repair (NER) pathways; defects in these pathways lead to a genetic instability known as xeroderma pigmentosum (XP) eventually developing skin cancers. We discovered that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. Here, we showed that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV through XPD and rpS3 interaction thus augmenting the helicase activity of XPD protein. Additionally, the knockdown of rpS3 caused reduced NER efficiency and the subsequent addition of rpS3 restored the helicase activity of the TFIIH complex in cells. We present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We also propose that rpS3 is a novel accessory protein of the NER pathway and its recruitment to the repair machinery upon UV damage augments repair efficiency by enhancing XPD helicase function and increasing its turnover rate, thus facilitating DNA repair. In addition to this, manipulations of rpS3 gene could complement XP-D phenotype and prevent UV damage and skin cancer occurrence. Animal studies strongly indicate that these result could be applied not only for the development of cancer drugs but also for cosmeceuticals. [1] FASEB J. (2020) 34: 8102 [2] Cell &amp; Mol Life Sci (2021) 78: 3591 Citation Format: Joon Kim, Yong-Joon Park, Tae Sung Kim, Woo Sung Ahn, Hag Dong Kim. RPS3 positively regulates nucleotide excision repair with respect to XP-D and skin cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3747.

Transcription of ncRNAs promotes repair of UV induced DNA lesions in Saccharomyces cerevisiae subtelomeres.

Ultraviolet light causes DNA lesions that are removed by nucleotide excision repair (NER). The efficiency of NER is conditional to transcription and chromatin structure. UV induced photoproducts are repaired faster in the gene transcribed strands than in the non-transcribed strands or in transcriptionally inactive regions of the genome. This specificity of NER is known as transcription-coupled repair (TCR). The discovery of pervasive non-coding RNA transcription (ncRNA) advocates for ubiquitous contribution of TCR to the repair of UV photoproducts, beyond the repair of active gene-transcribed strands. Chromatin rules transcription, and telomeres form a complex structure of proteins that silences nearby engineered ectopic genes. The essential protective function of telomeres also includes preventing unwanted repair of double-strand breaks. Thus, telomeres were thought to be transcriptionally inert, but more recently, ncRNA transcription was found to initiate in subtelomeric regions. On the other hand, induced DNA lesions like the UV photoproducts must be recognized and repaired also at the ends of chromosomes. In this study, repair of UV induced DNA lesions was analyzed in the subtelomeric regions of budding yeast. The T4-endonuclease V nicking-activity at cyclobutene pyrimidine dimer (CPD) sites was exploited to monitor CPD formation and repair. The presence of two photoproducts, CPDs and pyrimidine (6,4)-pyrimidones (6-4PPs), was verified by the effective and precise blockage of Taq DNA polymerase at these sites. The results indicate that UV photoproducts in silenced heterochromatin are slowly repaired, but that ncRNA transcription enhances NER throughout one subtelomeric element, called Y', and in distinct short segments of the second, more conserved element, called X. Therefore, ncRNA-transcription dependent TCR assists global genome repair to remove CPDs and 6-4PPs from subtelomeric DNA.

TREX1 degrades the 3' end of the small DNA oligonucleotide products of nucleotide excision repair in human cells.

The nucleotide excision repair (NER) machinery removes UV photoproducts from DNA in the form of small, excised damage-containing DNA oligonucleotides (sedDNAs) ∼30 nt in length. How cells process and degrade these byproducts of DNA repair is not known. Using a small scale RNA interference screen in UV-irradiated human cells, we identified TREX1 as a major regulator of sedDNA abundance. Knockdown of TREX1 increased the level of sedDNAs containing the two major UV photoproducts and their association with the NER proteins TFIIH and RPA. Overexpression of wild-type but not nuclease-inactive TREX1 significantly diminished sedDNA levels, and studies with purified recombinant TREX1 showed that the enzyme efficiently degrades DNA located 3′ of the UV photoproduct in the sedDNA. Knockdown or overexpression of TREX1 did not impact the overall rate of UV photoproduct removal from genomic DNA or cell survival, which indicates that TREX1 function in sedDNA degradation does not impact NER efficiency. Taken together, these results indicate a previously unknown role for TREX1 in promoting the degradation of the sedDNA products of the repair reaction. Because TREX1 mutations and inefficient DNA degradation impact inflammatory and immune signaling pathways, the regulation of sedDNA degradation by TREX1 may contribute to photosensitive skin disorders.

Ribosomal protein S3 associates with the TFIIH complex and positively regulates nucleotide excision repair.

In mammalian cells, the bulky DNA adducts caused by ultraviolet radiation are mainly repaired via the nucleotide excision repair (NER) pathway; some defects in this pathway lead to a genetic disorder known as xeroderma pigmentosum (XP). Ribosomal protein S3 (rpS3), a constituent of the 40S ribosomal subunit, is a multi-functional protein with various extra-ribosomal functions, including a role in the cellular stress response and DNA repair-related activities. We report that rpS3 associates with transcription factor IIH (TFIIH) via an interaction with the xeroderma pigmentosum complementation group D (XPD) protein and complements its function in the NER pathway. For optimal repair of UV-induced duplex DNA lesions, the strong helicase activity of the TFIIH complex is required for unwinding damaged DNA around the lesion. Here, we show that XP-D cells overexpressing rpS3 showed markedly increased resistance to UV radiation through XPD and rpS3 interaction. Additionally, the knockdown of rpS3 caused reduced NER efficiency in HeLa cells and the overexpression of rpS3 partially restored helicase activity of the TFIIH complex of XP-D cells in vitro. We also present data suggesting that rpS3 is involved in post-excision processing in NER, assisting TFIIH in expediting the repair process by increasing its turnover rate when DNA is damaged. We propose that rpS3 is an accessory protein of the NER pathway and its recruitment to the repair machinery augments repair efficiency upon UV damage by enhancing XPD helicase function and increasing its turnover rate.

A Method for Assessing the Efficiency of the Nucleotide Excision Repair System Ex Vivo.

The nucleotide excision repair (NER) is one of the main repair systems present in the cells of living organisms. It is responsible for the removal of a wide range of bulky DNA lesions. We succeeded in developing a method for assessing the efficiency of NER in the cell (ex vivo), which is a method based on the recovery of TagRFP fluorescent protein production through repair of the damage that blocks the expression of the appropriate gene. Our constructed plasmids containing bulky nFlu or nAnt lesions near the tagrfp gene promoter were shown to undergo repair in eukaryotic cells (HEK 293T) and that they can be used to analyze the efficiency of NER ex vivo. A comparative analysis of the time dependence of fluorescent cells accumulation after transfection with nFlu- and nAnt-DNA revealed that there are differences in how efficient their repair by the NER system of HEK 293T cells can be. The method can be used to assess the cell repair status and the repair efficiency of different structural damages.  

Bacterial Chromosome Replication and DNA Repair During the Stringent Response.

The stringent response regulates bacterial growth rate and is important for cell survival under changing environmental conditions. The effect of the stringent response is pleiotropic, affecting almost all biological processes in the cell including transcriptional downregulation of genes involved in stable RNA synthesis, DNA replication, and metabolic pathways, as well as the upregulation of stress-related genes. In this Review, we discuss how the stringent response affects chromosome replication and DNA repair activities in bacteria. Importantly, we address how accumulation of (p)ppGpp during the stringent response shuts down chromosome replication using highly different strategies in the evolutionary distant Gram-negative Escherichia coli and Gram-positive Bacillus subtilis. Interestingly, (p)ppGpp-mediated replication inhibition occurs downstream of the origin in B. subtilis, whereas replication inhibition in E. coli takes place at the initiation level, suggesting that stringent cell cycle arrest acts at different phases of the replication cycle between E. coli and B. subtilis. Furthermore, we address the role of (p)ppGpp in facilitating DNA repair activities and cell survival during exposure to UV and other DNA damaging agents. In particular, (p)ppGpp seems to stimulate the efficiency of nucleotide excision repair (NER)-dependent repair of DNA lesions. Finally, we discuss whether (p)ppGpp-mediated cell survival during DNA damage is related to the ability of (p)ppGpp accumulation to inhibit chromosome replication.

Enhanced Activity of Variant DNA Polymerase β (D160G) Contributes to Cisplatin Therapy by Impeding the Efficiency of NER.

Cisplatin, commonly used in a variety of cancer treatments, induces apoptosis in cancer cells by causing lethal DNA damage. Several DNA repair pathways participate in regulation of cisplatin treatment, leading to cisplatin sensitivity or resistance in cancer cells. DNA polymerase β (pol β), a key protein involved in base excision repair, confers a response to cisplatin therapy that is dependent on polymerase activity. Pol β D160G mutation with enhanced polymerase activity, previously identified in clear cell renal cell carcinoma, enhances the sensitivity of human cancer cells and mouse xenografts to cisplatin by limiting the efficiency of nucleotide excision repair (NER). Notably, the D160G mutation impedes the recruitment of XPA to cisplatin-induced sites of DNA damage, leading to unrepaired damage and further inducing cell death. Molecular architecture analysis indicated that the D160G mutation alters protein-DNA interactions and the surface electrostatic properties of the DNA-binding regions, resulting in greater DNA affinity and polymerase activity compared with wild-type pol β. Collectively, these results indicate that enhancing pol β activity impedes the efficiency of NER and provide a promising adjuvant therapeutic strategy for cisplatin chemotherapy. IMPLICATIONS: Our studies demonstrate that polβ D160G mutation with enhanced polymerase activity impedes NER efficiency during the repair of cisplatin-induced DNA damage, leading to increased cisplatin sensitivity in cancer cells.

Nucleotide Excision Repair and Impact of Site-Specific 5',8-Cyclopurine and Bulky DNA Lesions on the Physical Properties of Nucleosomes.

The nonbulky 5',8-cyclopurine DNA lesions (cP) and the bulky, benzo[ a]pyrene diol epoxide-derived stereoisomeric cis- and trans- N2-guanine adducts (BPDE-dG) are good substrates of the human nucleotide excision repair (NER) mechanism. These DNA lesions were embedded at the In or Out rotational settings near the dyad axis in nucleosome core particles reconstituted either with native histones extracted from HeLa cells (HeLa-NCP) or with recombinant histones (Rec-NCP). The cP lesions are completely resistant to NER in human HeLa cell extracts. The BPDE-dG adducts are also NER-resistant in Rec-NCPs but are good substrates of NER in HeLa-NCPs. The four BPDE-dG adduct samples are excised with different efficiencies in free DNA, but in HeLa-NCPs, the efficiencies are reduced by a common factor of 2.2 ± 0.2 relative to the NER efficiencies in free DNA. The NER response of the BPDE-dG adducts in HeLa-NCPs is not directly correlated with the observed differences in the thermodynamic destabilization of HeLa-NCPs, the Förster resonance energy transfer values, or hydroxyl radical footprint patterns and is weakly dependent on the rotational settings. These and other observations suggest that NER is initiated by the binding of the DNA damage-sensing NER factor XPC-RAD23B to a transiently opened BPDE-modified DNA sequence that corresponds to the known footprint of XPC-DNA-RAD23B complexes (≥30 bp). These observations are consistent with the hypothesis that post-translational modifications and the dimensions and properties of the DNA lesions are the major factors that have an impact on the dynamics and initiation of NER in nucleosomes.

A role for Saccharomyces cerevisiae Centrin (Cdc31) in mitochondrial function and biogenesis.

Centrins belong to a family of proteins containing calcium-binding EF-hand motifs that perform well-established roles in centrosome and spindle pole body (SPB) duplication. Yeast encodes a single Centrin protein (Cdc31) that binds components in the SPB. However, further studies revealed a role for Centrins in mRNA export, and interactions with contractile filaments and photoreceptors. In addition, human Centrin-2 can bind the DNA-lesion recognition factor XPC, and improve the efficiency of nucleotide excision repair. Similarly, we reported that yeast Cdc31 binds Rad4, a functional counterpart of the XPC DNA repair protein. We also found that Cdc31 is involved in the ubiquitin/proteasome system, and mutations interfere with intracellular protein turnover. In this report, we describe new findings that indicate a role for Cdc31 in the energy metabolism pathway. Cdc31 and cdc31 mutant proteins showed distinct interactions with proteins in energy metabolism, and mutants showed sensitivity to oxidative stress and poor growth on non-fermentable carbon. Significant alteration in mitochondrial morphology was also detected. Although it is unclear how Cdc31 contributes to so many unrelated mechanisms, we propose that by controlling SPB duplication Centrin proteins might link the cellular responses to DNA damage, oxidative load and proteotoxic stresses to growth control.

Lesion Sensing during Initial Binding by Yeast XPC/Rad4: Toward Predicting Resistance to Nucleotide Excision Repair.

Nucleotide excision repair (NER) excises a variety of environmentally derived DNA lesions. However, NER efficiencies for structurally different DNA lesions can vary by orders of magnitude; yet the origin of this variance is poorly understood. Our goal is to develop computational strategies that predict and identify the most hazardous, repair-resistant lesions from the plethora of such adducts. In the present work, we are focusing on lesion recognition by the xeroderma pigmentosum C protein complex (XPC), the first and required step for the subsequent assembly of factors needed to produce successful NER. We have performed molecular dynamics simulations to characterize the initial binding of Rad4, the yeast orthologue of human XPC, to a library of 10 different lesion-containing DNA duplexes derived from environmental carcinogens. These vary in lesion chemical structures and conformations in duplex DNA and exhibit a wide range of relative NER efficiencies from repair resistant to highly susceptible. We have determined a promising set of structural descriptors that characterize initial binding of Rad4 to lesions that are resistant to NER. Key initial binding requirements for successful recognition are absent in the repair-resistant cases: There is little or no duplex unwinding, very limited interaction between the β-hairpin domain 2 of Rad4 and the minor groove of the lesion-containing duplex, and no conformational capture of a base on the lesion partner strand. By contrast, these key binding features are present to different degrees in NER susceptible lesions and correlate to their relative NER efficiencies. Furthermore, we have gained molecular understanding of Rad4 initial binding as determined by the lesion structures in duplex DNA and how the initial binding relates to the repair efficiencies. The development of a computational strategy for identifying NER-resistant lesions is grounded in this molecular understanding of the lesion recognition mechanism.

Melanoma cells resistant towards MAPK inhibitors exhibit reduced TAp73 expression mediating enhanced sensitivity to platinum-based drugs

The efficacy of targeted MAPK signalling pathway inhibitors (MAPKi) in metastatic melanoma therapy is limited by the development of resistance mechanisms that results in disease relapse. This situation still requires treatment alternatives for melanoma patients with acquired resistance to targeted therapy. We found that melanoma cells, which developed resistance towards MAPKi show an enhanced susceptibility to platinum-based drugs, such as cisplatin and carboplatin. We found that this enhanced susceptibility inversely correlates with the expression level of the p53 family member TAp73. We show that the lower expression of the TAp73 isoform in MAPKi-resistant melanoma cells enhances accumulation of DNA double-strand breaks upon cisplatin and carboplatin treatment by reducing the efficiency of nucleotide excision repair. These data suggest that a subgroup of melanoma patients with acquired resistance to MAPKi treatment and low TAp73 expression can benefit from chemotherapy with platinum-based drugs as a second-line therapy.

Abstract 4155: USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer

Abstract UV radiation is a major risk factor for skin cancer, the most common cancer in the United States. UV exposure leads to DNA damage by formation of dimers between adjacent pyrimidine bases. The nucleotide excision repair (NER) process is responsible for removal of UV-induced DNA damage. When the damage is left unrepaired due to inefficient NER in disorders like xeroderma pigmentosum, it leads to skin tumorigenesis. Although the main players of the NER pathway have been characterized, the molecular regulation of the pathway is less understood. Understanding the regulation, particularly post-translational regulation, of the NER pathway has the potential to yield modulators of NER efficiency to prevent skin cancer. We found that chronic UV irradiation led to down-regulation of ubiquitin specific peptidase 11 (USP11) in mouse skin tissue. We also found that USP11 is decreased in skin tumors in mice and humans. Moreover, high-throughput proteomics analysis had predicted that USP11 may interact with NER factor XPC. Hence, we undertook this study to determine the role of USP11 in NER and cellular response to UV. We found that USP11 promoted the NER pathway by regulating XPC activity. USP11 promoted XPC deubiquitination and retention at the DNA damage sites. We also found that USP11 interaction with ubiquitinated XPC was enhanced post-UV irradiation, and that USP11 was recruited to the chromatin post-UV damage. Further, squamous cell carcinoma cells showed reduced survival with inhibition of USP11 after UV exposure. Our findings indicate that USP11 plays an important role in maintaining NER capacity, and suggest that USP11 acts as a tumor suppressor via its role in DNA repair. Citation Format: Palak Shah, Lei Qiang, Seungwon Yang, Keyoumars Soltani, Yu-Ying He. USP11 regulates UV-induced DNA damage repair and cell survival associated with skin cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4155.

Enhancement of UVB-induced DNA damage repair after a chronic low-dose UVB pre-stimulation

Absorption of solar ultraviolet (UV) radiation by DNA leads to the formation of the highly mutagenic cyclobutane pyrimidine dimer (CPD). The mutagenicity of CPD is caused, in part, by the fact that their recognition and repair by the nucleotide excision repair (NER) pathway is challenging and slow. It has been previously shown that a pre-stimulation with genotoxic agents improve NER efficiency of CPD, indicating a potential adaptive response of this repair pathway. We have pre-treated human dermal fibroblasts with repeated subletal low doses of UVB (chronic low-dose of UVB; CLUV) to determine whether it could enhance NER capacity to repair CPD. Our results show that CLUV pre-treatment greatly enhances CPD repair but have little effect on the repair of another UV-induced bypirimidine photoproduct, the pyrimidine (6-4) pyrimidone photoproducts (6-4 PP). We have determined that the CLUV treatment activates p53 and we found an increase of DDB2 and XPC gene expression. This is consistent with an increasing level of NER recognition proteins, DDB2 and XPC, we found concentrated at the chromatin. This study represents the first demonstration that chronic UVB exposure can stimulate NER pathway. Altogether, these results shed light on the potential adaptability of the NER by chronic UVB irradiation and the mechanisms involved.

Nucleotide excision repair is a potential therapeutic target in multiple myeloma

Despite the development of novel drugs, alkylating agents remain an important component of therapy in multiple myeloma (MM). DNA repair processes contribute towards sensitivity to alkylating agents and therefore we here evaluate the role of nucleotide excision repair (NER), which is involved in the removal of bulky adducts and DNA crosslinks in MM. We first evaluated NER activity using a novel functional assay and observed a heterogeneous NER efficiency in MM cell lines and patient samples. Using next-generation sequencing data, we identified that expression of the canonical NER gene, excision repair cross-complementation group 3 (ERCC3), significantly impacted the outcome in newly diagnosed MM patients treated with alkylating agents. Next, using small RNA interference, stable knockdown and overexpression, and small-molecule inhibitors targeting xeroderma pigmentosum complementation group B (XPB), the DNA helicase encoded by ERCC3, we demonstrate that NER inhibition significantly increases sensitivity and overcomes resistance to alkylating agents in MM. Moreover, inhibiting XPB leads to the dual inhibition of NER and transcription and is particularly efficient in myeloma cells. Altogether, we show that NER impacts alkylating agents sensitivity in myeloma cells and identify ERCC3 as a potential therapeutic target in MM.

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway.

The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH., (2015) Biochemistry, 54( (34), ), 5263−726270861]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.