--- arxiv_id: PMC12909189 title: "Studies on the functionality of the TC-NER ERCC6-M1097V protein variant frequently found in Louisiana patients with PCa upon UV damage." authors: - Ogundepo O - De Benedetti A difficulty: Advanced tags: - Oncology - Rare Disease published_at: 2025 flecto_url: https://flecto.zer0ai.dev/papers/PMC12909189/ lang: en --- ## Head Title ### ERCC6-M1097V Variant in TC-NER: UV Damage Response in Louisiana PCa Patients ## Hero H1 Studies on the Functionality of the TC-NER ERCC6-M1097V Protein Variant Frequently Found in Louisiana Patients with PCa upon UV Damage ## Hero Authors ### Oluwatobi Ogundepo & Arrigo De Benedetti* ## Hero Affiliation Department of Biochemistry and Molecular Biology, Louisiana State University Health Shreveport, LA, United States ## Hero Meta ### Published February 3, 2026 · DOI: 10.3389/fonc.2025.1679379 ## Hero Button ### Read on PMC ↗ ## Abstract Heading ### Abstract ## Abstract Body ERCC6 , also known as CSB (Cockayne Syndrome B) , is a key protein involved in transcription-coupled nucleotide excision repair (TC-NER) , a DNA repair process that removes lesions blocking RNA polymerase. ERCC6 has multifaceted roles including chromatin remodeling, transcription regulation, oxidative stress response, and coordination with other DNA repair proteins. Mutations in ERCC6 lead to Cockayne Syndrome and other neurodegenerative disorders, but some variants, such as M1097V , have been associated with cancer risk, particularly prostate cancer (PCa) in African Americans (AAs) in Louisiana . Recent studies have explored the functional impact of ERCC6 variants in PCa, especially among AAs, who face higher incidence and more aggressive forms of the disease. A notable finding is that the M1097V variant increases cellular tolerance to UV damage , suggesting not only a possible evolutionary benefit but also a potential risk for mutagenesis when exposed to complex environmental carcinogens. Given the high mutation burden in mismatch repair (MMR) and NER genes observed in AA patients with PCa, a synthetic lethality strategy targeting both TC-NER and homologous recombination repair (HRR) pathways could be effective, including combining agents like CDDP (cisplatin) with inhibitors of RAD54, such as J54 . ## Introduction Heading ### Introduction ## Introduction Stat Label Aa ### M1097V frequency in African American PCa patients ## Introduction Stat Label Cc ### M1097V frequency in Caucasian PCa patients ## Introduction P1 Prostate cancer (PCa) is the most commonly diagnosed non-cutaneous malignancy and the second-leading cause of cancer-related death among men in the United States. African American (AA) men bear a disproportionate burden of this disease compared to men of European ancestry, exhibiting higher incidence rates, earlier onset, and increased mortality from treatment-refractory disease. While socioeconomic and healthcare access disparities contribute to these differences, accumulating evidence suggests that biological factors, including genomic and molecular alterations, may play a critical role in driving the aggressive phenotype observed in AA patients. ## Introduction P2 The DNA damage response and repair pathways (DDRR), collectively known as the "repairome," are essential for maintaining genomic stability and preventing malignant transformation. In PCa, alterations in key DNA repair genes—such as BRCA1 , BRCA2 , ATM , and MLH1 —have been associated with tumor progression, poor prognosis, and sensitivity to targeted therapies, including PARP inhibitors and platinum-based agents. The success of PARPi for metastatic castration-resistant prostate cancer (mCRPC) is defining new treatment options, and while cisplatin-based therapy is not currently the treatment of choice for PCa, current trends suggest that lower dosing combined with DNA repair inhibitors could be quite effective . ## Introduction Concept Card Heading ### ERCC6/CSB: A Multifunctional DNA Repair Protein ## Introduction Concept Li1 Primary function: Mediates the dislodging of transcription Elongation Complexes stuck at DNA-distorting lesions ## Introduction Concept Li2 ### Anti-apoptotic factor: Tips the cell towards proliferation and survival ## Introduction Concept Li3 Loss of function: Results in cell cycle arrest, senescence via p53 interaction, and Cockayne syndrome (progeria) ## Introduction Concept Li4 M1097V variant: Found at 21% frequency in AA-PCa (vs 1% in Caucasians), identified as a significant risk factor in meta-analyses of multiple cancer types worldwide ## Introduction Aim Heading ### Study Objectives ## Introduction Aim Body In this study, the M1097V genomic mutation was introduced via CRISPR/Cas9 in a panel of common PCa cell lines, including PCa2 cells derived from an AA patient. The consequences for the repair of lesions requiring TC-NER (UV and cisplatin resistance) were investigated as a first assessment of how this mutant protein might interact with environmental exposures. In Louisiana, PCa disparity is far more prevalent, with higher incidence and worse overall survival attributed to both genetic components and dietary habits, compounded by much greater health risks from a regional toxic environment. ## Methods Heading ### Materials & Methods ## Methods Card1 Heading ### CRISPR/Cas9 Site-Directed Mutagenesis ## Methods Card1 Body Guide RNA, donor DNA, and TrueCut Cas9 protein were designed using ThermoFisher TrueDesign. Cells were transfected at 70% confluency using CRISPRMAX reagent in Opti-MEM. After 48 hours, single-cell clones were created and screened. ## Methods Card2 Heading ### Plasmid Vector SDM ## Methods Card2 Body The M1097V mutation was introduced in OriGene expression vector RC219020 using the QuickChangeII SDM kit. Transient transfections were carried out with Lipofectamine-3000 for 48 hours. ## Methods Card3 Heading ### Gel Electrophoresis ## Methods Card3 Body 1% agarose gels with EtBr at 80V. PCR-RFLP with Hin1II (NlaIII) for clone verification. Imaged with BIORAD ChemiDoc system, followed by confirmatory sequencing. ## Methods Card4 Heading ### Dot Blot (DNA Southwestern Blot) ## Methods Card4 Body 50,000 cells plated, UV-exposed for 30 seconds, then recovered at different time points. Lysed, dot blotted, and probed with anti-CPD antibody to detect cyclobutane pyrimidine dimers. ## Methods Card5 Heading ### Proliferation Assay ## Methods Card5 Body Cells seeded in 96-well plates at 50% confluency, exposed to various UV doses. Growth monitored via IncuCyte S3 with phase contrast imaging every 4 hours. ## Methods Card6 Heading ### ATPase Assay ## Methods Card6 Body Performed with ADP-Hunter using ~50 ng of immunopurified ERCC6 and ±50 ng plasmid DNA. Kinetics measured at steady state after 15-minute pre-incubation at 37°C. ## Methods Card7 Heading ### Statistical Analysis ## Methods Card7 Body GraphPad Prism 9 for statistical analysis. Results as mean ± SEM. Student's t-test for two-group comparisons. Significance: *p < 0.05, **p < 0.01, ***p < 0.001. ## Results Figure1 Heading ### ERCC6 Protein Structure & Conservation ## Results Figure1 Text ERCC6 is an essential, highly conserved gene from yeast to mammals. Given its essentiality, mutations are rare and almost absent from the PCa TCGA-500 database. Therefore, the unusual frequency of the M1097V variant , especially in African Americans, may be a peculiarity of the Louisiana population. Starting from the genomic site-directed mutagenesis work, the M1097V mutations were introduced in multiple cell lines via CRISPR-mediated recombination, yielding hetero- and homozygous (bi-allelic) mutants. ## Results Figure1 Figcaption Figure 1: (A) ERCC6 protein domain structure showing N-terminal, ATPase binding domain, helicase C-terminal domain, and location of M1097V mutation. (B) Evolutionary conservation of ERCC6 across species (human, mouse, plant, yeast). (C) Schematic of ERCC6 functional regions including N-CSB, M-CSB, and C-CSB domains. ## Results Crispr Heading ### CRISPR/Cas9 M1097V Knock-In ## Results Crispr Text As there are no available cell lines carrying the M1097V mutation, CRISPR-mediated editing was used to introduce this variant. The M1097V knock-in was generated and validated in multiple prostate cancer cell lines including C4-2B, DU145, PC3, and PCa2 (an AA patient-derived line). Both heterozygous and homozygous (bi-allelic) clones were obtained and confirmed by PCR-RFLP and sequencing. ## Results Crispr Table Caption ### Table 1: Primers and CRISPR components used for M1097V knock-in ## Results Crispr Figcaption Figure 2: Generation and validation of M1097V knock-in using CRISPR/Cas9. (A) Schematic of M1097 vs M1097V nucleotide and amino acid sequences. (B) CRISPR/Cas9 editing workflow with homology-directed repair. (C) PCR amplification and Hin1II cleavage. (D) Plasmid confirmation from OriGene. (E) Clone confirmation via PCR-RFLP across cell lines. ## Results Uv Heading ### UV Sensitivity & CPD Repair ## Results Uv Key Finding Heading ### Key Finding ## Results Uv Key Finding Body Against the initial hypothesis, the M1097V mutation conferred greater resistance to UV doses and faster resolution of UV-induced CPDs . An "overactive" TC-NER mechanism can be more mutagenic under the right conditions than an underactive, deficient one—the faster repair paradoxically increases the chance of introducing mismatches during strand replacement. ## Results Uv P1 UV and CDDP sensitivity were assessed in the ERCC6-mutant derivative clones. Surprisingly, the M1097V mutation conferred somewhat greater resistance to UV doses and faster resolution of UV-induced cyclobutane pyrimidine dimers (CPDs). Interestingly, the PCa2 line from an AA patient , which carries a different ERCC6 mutation (Y776C), also showed remarkable activity in CPD removal compared to all other lines. This UV resistance clearly depends on ERCC6, as siRNA knockdown drastically reduced UV survival . Control NT1-Nek1-KO cells showed almost no DNA repair (CPD removal) even after one full day. ## Results Uv P2 During NER strand replacement at the incision site, there is a significant chance of introducing mismatches if the bulky lesions are elevated, particularly in the presence of 8-oxoguanine (8OG). Mismatch repair (MMR) can actually be more mutagenic than NER-mediated correction of bulky lesions due to the lack of precise strand discrimination. Hence, UV sensitivity (orchestrated via CPD removal) and CDDP sensitivity (via complex combination pathways) are not overlapping as one might expect. ## Results Uv Fig3 Caption Figure 3: UV sensitivity proliferation assays. (A, B) Confluency changes in DU145-G3 and PC3-A8 cells after 0–15 seconds UV exposure. (C, D) Growth rate comparisons (ΔConfluency/Time) showing statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001). ## Results Uv Fig4 Caption Figure 4: Dot blot (DNA Southwestern blot) showing CPD removal kinetics. Time points: −UV, 0, 6, 12, 18, and 24 hours post-UV. M1097V mutants (D3, A8, G3) and PCa2 demonstrate faster CPD removal compared to wild-type and NT1-Nek1KO controls. ## Results Uv Fig5 Caption Figure 5: ERCC6 dependency validation. (A–D) Proliferation assays of parental cells treated with ERCC6 siRNA and exposed to 5s UV, showing that ERCC6 knockdown drastically reduces UV survival. (E) Western blot confirming ERCC6 protein knockdown at various siRNA concentrations. ## Results Cisplatin Heading ### Cisplatin Sensitivity ## Results Cisplatin Text Unlike the UV resistance phenotype, the response to cisplatin (CDDP) showed a different pattern. UV sensitivity (orchestrated via CPD removal) and CDDP sensitivity (via complex combination pathways including NER and HRR) are not overlapping mechanisms . CSB may be beneficial in UV damage repair but not necessarily in cisplatin-induced damage. Importantly, this means that the ERCC6 variants found in AA-PCa can still be targeted by an NER and HRR combination strategy, as these are expected to yield more double-strand breaks (DSBs) during cisplatin inter-/intrastrand crosslinks (CDDP-ISLs) processing. ## Results Cisplatin Fig6 Caption Figure 6: Cisplatin (CDDP) sensitivity assays. (A–D) Proliferation of DU145, G3, PC3, and A8 cells pretreated with 1, 5, and 15 µg/mL cisplatin for 6 hours, then monitored over several days. The data show that cisplatin sensitivity is mechanistically distinct from UV sensitivity. ## Results Atpase Heading ### ATPase Activity ## Results Atpase Text ATPase activity of purified ERCC6 wild-type and M1097V mutant was compared using the ADP-Hunter assay. The M1097V variant showed subtle differences in DNA-dependent ATPase kinetics. These complex ATPase cycles—some DNA-independent—promote large structural rearrangements while displacing the stuck transcription elongation complex. Detailed biochemical studies, similar to those done with the E. coli ERCC6 paralog Mfd, will be needed to fully characterize the impact of this mutation on enzymatic function. ## Results Atpase Fig7 Caption Figure 7: ATPase activity of purified ERCC6. Left: ATPase kinetics (RLU vs. time) for wild-type and M1097V, with and without DNA. Right: Western blot confirming purified protein (Mock IP, CSB-wt-Flag, CSB-M1097V-Flag). ## Discussion Heading ### Discussion ## Discussion Roles Heading ### Multifaceted Roles of ERCC6/CSB ## Discussion Role1 TC-NER: Detects and initiates repair of DNA lesions blocking transcription. When RNA Pol II stalls at UV-induced CPDs, ERCC6 recruits repair factors to remove the lesion and resume transcription. ## Discussion Role2 Chromatin Remodeling: Possesses ATP-dependent chromatin remodeling activity, crucial for providing repair machinery access to DNA in compact chromatin regions. ## Discussion Role3 Transcription Regulation: Regulates gene expression by interacting with transcription machinery, influencing RNA Pol I and Pol II pausing and restart. ## Discussion Role4 Repair Coordination: Interacts with TC-NER factors including CSA (ERCC8), XPG, TFIIH, and UVSSA to coordinate the repair process. ## Discussion Role5 Oxidative Damage: Implicated in repair of oxidative DNA damage, helping maintain mitochondrial function and cellular redox balance under stress. ## Discussion Role6 Disease Relevance: Mutations cause Cockayne Syndrome, a rare autosomal recessive disorder with growth failure, neurodegeneration, and premature aging. ## Discussion M1097V Heading ### M1097V: From Cancer Risk to Therapeutic Target ## Discussion M1097V Text While loss-of-function mutations cause severe syndromes, most missense variants’ functions have not been well studied. The M1097V polymorphism has been identified as a significant risk factor for cancer in meta-analyses of several cancer types worldwide, and was recently noted for its unusually high prevalence in AA-PCa (21% vs 1% in Caucasians). The faster CPD repair activity in M1097V mutants forced a rethinking of the initial hypothesis: an "overactive" TC-NER mechanism can paradoxically be more mutagenic under certain conditions because of increased mismatches during rapid strand replacement. ## Discussion Strategy Heading ### Synthetic Lethality Strategy: CDDP + J54 ## Discussion Strategy Body The ERCC6 variants found in AA-PCa can still be successfully targeted by an NER and HRR combination strategy —for example, with a synthetic lethal combination of cisplatin (CDDP) and J54 (a RAD54 inhibitor). These combinations are expected to yield more double-strand breaks during cisplatin crosslink processing, offering an effective therapeutic approach even against cells with enhanced TC-NER activity. ## Discussion Therapeutic Heading ### Therapeutic Implications & Future Directions ## Discussion Therapeutic P1 AAs are at higher risk for developing PCa and have more aggressive, treatment-refractory disease. Since most PCa therapies focus on AR signaling inhibition, combination approaches targeting DNA repair could bypass or eliminate the need for androgen deprivation therapy (ADT) and its significant side effects. This is especially relevant for neuroendocrine prostate cancer (NEPC) cases, which are more common in AA patients and do not respond to ADT/ARSI. ## Discussion Therapeutic P2 Endogenous ERCC6 expression is quite low and further complicated by the ERCC6-PGBD3 transposon fusion gene. Future studies will include the S636N mutation (also found only in Louisiana PCa), more detailed ATPase analyses, and investigation of the complex biochemical cycles underlying ERCC6-mediated transcription complex displacement. ## Conclusions Heading ### Conclusions ## Conclusions Body This study underscores the critical and multifaceted role of ERCC6/CSB in DNA repair and transcription regulation, particularly through the TC-NER pathway. The M1097V variant enhances UV resistance in PCa cells, suggesting a possible evolutionary adaptation with modern therapeutic implications . This variant, along with other ERCC6 alterations found predominantly in AA patients with PCa, may explain differences in treatment response. Synthetic lethality approaches targeting both NER and HRR pathways (e.g., CDDP + J54) could offer personalized alternatives to androgen deprivation therapy, representing a promising direction for addressing PCa disparities. ## Supplementary Heading ### Additional Information ## Supplementary Author Heading ### Author Contributions ## Supplementary Author Body OO: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing, Writing – original draft. AD: Conceptualization, Formal Analysis, Funding acquisition, Investigation, Project administration, Supervision, Validation, Writing – original draft, Writing – review & editing. ## Supplementary Funding Heading ### Funding ## Supplementary Funding Body ### Chancellor Award 2024 to AB. ## Supplementary Ack Heading ### Acknowledgments ## Supplementary Ack Body The authors thank the INLET facility of LSU Health Shreveport (RRID: SCR_024775), especially Ana Maria Dragoi and Brian Latimer for their assistance in working with the IncuCyte. ## Supplementary Conflict Heading ### Conflict of Interest ## Supplementary Conflict Body The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. ## Supplementary Data Heading ### Data Availability ## Supplementary Data Body The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. ## References Heading ### References (35) ## Footer This article is an accessible summary generated by Flecto. Please refer to the original publication for the authoritative version.