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Background: Cisplatin (CDDP) remains a key agent in the treatment of muscle-infiltrating bladder carcinoma (MIBC). However, a proportion of MIBC patients do not respond to chemotherapy, which may be caused by the increased repair of CDDP-induced DNA damage. The purpose of this study was to explore the prognostic value of proteins involved in nucleotide excision repair (NER) and translesion DNA synthesis (TLS) in MIBC patients. Methods: This is a retrospective analysis of 86 MIBC patients. The XPA, XPF, XPG, ERCC1, POLI, POLH and REV3L proteins were stained in primary bladder tumors and their levels were analyzed both in the total cohort and in a subgroup with metastatic urothelial carcinoma (mUC) that received gemcitabine and CDDP as a first-line therapy. Both cohorts were divided by percentage of cancer cells stained positive for each protein into subgroups with high and low expression. In the same manner, the combined expression of NER (XPA + ERCC1 + XPF + XPG) and TLS (POLI + POLH + REV3L), as the whole pathways, was analyzed. Results: Mortality was 89.5% at the median follow-up of 120.2 months. In the total cohort, patients with tumors stained positive for XPA, XPG and POLI had significantly worse overall survival (OS) compared to those with negative staining [hazard ratio (HR) = 0.60, 0.62 and 0.53, respectively]. Both XPG and POLI were independent prognostic factors in multivariate analyses (MVA). In addition, an increase in NER and TLS pathway expression was significantly associated with worse OS in the total cohort (HR = 0.54 and 0.60, respectively). In the mUC subgroup, high POLI expression was associated with significant deterioration of OS (HR = 0.56) in univariate analyses, and its independent prognostic value was shown in MVA. Conclusions: Our study showed significant correlations between the tumor expression of XPG and POLI, as well as NER and TLS as the whole pathways, and inferior OS. Hence, they could constitute prognostic biomarkers and potentially promising therapeutic targets in MIBC. However, a prospective trial is required for further validation, thereby overcoming the limitations of this study. [ABSTRACT FROM AUTHOR]

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

Abstract Background Cisplatin (CDDP) remains a key agent in the treatment of muscle-infiltrating bladder carcinoma (MIBC). However, a proportion of MIBC patients do not respond to chemotherapy, which may be caused by the increased repair of CDDP-induced DNA damage. The purpose of this study was to explore the prognostic value of proteins involved in nucleotide excision repair (NER) and translesion DNA synthesis (TLS) in MIBC patients. Methods This is a retrospective analysis of 86 MIBC patients. The XPA, XPF, XPG, ERCC1, POLI, POLH and REV3L proteins were stained in primary bladder tumors and their levels were analyzed both in the total cohort and in a subgroup with metastatic urothelial carcinoma (mUC) that received gemcitabine and CDDP as a first-line therapy. Both cohorts were divided by percentage of cancer cells stained positive for each protein into subgroups with high and low expression. In the same manner, the combined expression of NER (XPA + ERCC1 + XPF + XPG) and TLS (POLI + POLH + REV3L), as the whole pathways, was analyzed. Results Mortality was 89.5% at the median follow-up of 120.2 months. In the total cohort, patients with tumors stained positive for XPA, XPG and POLI had significantly worse overall survival (OS) compared to those with negative staining [hazard ratio (HR) = 0.60, 0.62 and 0.53, respectively]. Both XPG and POLI were independent prognostic factors in multivariate analyses (MVA). In addition, an increase in NER and TLS pathway expression was significantly associated with worse OS in the total cohort (HR = 0.54 and 0.60, respectively). In the mUC subgroup, high POLI expression was associated with significant deterioration of OS (HR = 0.56) in univariate analyses, and its independent prognostic value was shown in MVA. Conclusions Our study showed significant correlations between the tumor expression of XPG and POLI, as well as NER and TLS as the whole pathways, and inferior OS. Hence, they could constitute prognostic biomarkers and potentially promising therapeutic targets in MIBC. However, a prospective trial is required for further validation, thereby overcoming the limitations of this study.

The nucleotide excision repair (NER) system is one of the main ways to protect organisms from DNA damage caused by endogenous and exogenous carcinogens. NER deficiency increases genome mutations, chromosomal aberrations, and cancer viability. However, the genetic association between Wilms tumor and NER pathway gene polymorphisms needs to be further validated. We assessed the associations between 19 NER gene polymorphisms and Wilms tumor susceptibility in 416 cases and 936 controls from East China via the TaqMan method. We found that xeroderma pigmentosum group D (XPD) rs238406 and rs13181 significantly decreased the risk of Wilms tumor [adjusted odds ratio (OR) = 0.59, 95% confidence interval (CI) = 0.46–0.75, p <.0001; adjusted OR = 0.63, 95% CI = 0.44–0.89, p =.009, respectively]. Furthermore, the rs751402 and rs2296147 polymorphisms in the xeroderma pigmentosum group G (XPG) gene were significantly correlated with an increased risk for Wilms tumor (adjusted OR = 1.47, 95% CI = 1.03–2.09, p =.034; adjusted OR = 2.14, 95% CI = 1.29–3.56, p =.003, respectively). Expression quantitative trait loci (eQTL) analysis revealed that these four polymorphisms may affect the expression of genes that are adjacent to XPD and XPG. Our study provides evidence that XPD and XPG gene polymorphisms are associated with Wilms tumor risk. Nonetheless, these findings should be confirmed in a larger sample size. [ABSTRACT FROM AUTHOR]

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N2-(ADP-ribos-1-yl)-2′-deoxyguanosine (N2-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N2-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N2-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N2-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N2-ADPR-dG induced by cabbage butterfly pierisin-1. [ABSTRACT FROM AUTHOR]

Background: Trichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2, a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown. Objective: The aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1. Methods: Cellular immune phenotype was investigated using multicolor flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients. Results: We investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harbored novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of g-H2AX levels, decreased proliferation activity and reduced cell viability following UV irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo. Conclusion: In summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired BCR-mediated B-cell activation and activation-induced gene transcription. [ABSTRACT FROM AUTHOR]

Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF–ERCC1 nuclease forming a damaged 5′-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5′-flap and different size gap to analyze possible repair factor–replication factor interactions. Ternary XPA–FEN1–DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA–FEN1 complex formation in the absence of DNA due to protein–protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA–XPA–FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA–FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1. [ABSTRACT FROM AUTHOR]

Hydrochlorothiazide (HCTZ) is a widely utilized diuretic for the treatment of hypertension. The photosensitivity of HCTZ has been recognized for six decades, with UVA being considered the primary culprit. However, the precise molecular mechanism of HCTZ sensitizing skin to UV radiation remains unknown. In this study, we demonstrate that HCTZ exacerbates UVB-induced photosensitivity in normal skin by disrupting the DNA damage response, a crucial network responsible for maintaining epidermal homeostasis. Here, we found that HCTZ aggravates UVB-induced mouse skin damage. Through transcriptomic and proteomic profiling, we have found that the cell cycle and p53 signaling pathway may contribute to the photosensitivity caused by HCTZ. In keratinocytes, HCTZ promotes the transition from G1 to S phase and inhibits the p53 signaling pathway after exposure to UV radiation. We have found that HCTZ enhances the accumulation of DNA damage induced by UVB and impairs nucleotide excision repair (NER), which is responsible for repairing UVB-induced DNA lesions, by inhibiting the expression of NER-related genes and shortening the duration of G1 phase. Furthermore, pharmacologically inducing G1 arrest eliminates HCTZ-induced accumulation of damaged DNA. These findings unveil an unknown mechanism through which HCTZ impairs NER and interferes with UVB-induced cell cycle arrest, ultimately leading to improper response towards DNA damage and increased skin sensitivity.

DNA repair pathways play a critical role in genome stability, but in eukaryotic cells, they must operate to repair DNA lesions in the compact and tangled environment of chromatin. Previous studies have shown that the packaging of DNA into nucleosomes, which form the basic building block of chromatin, has a profound impact on DNA repair. In this review, we discuss the principles and mechanisms governing DNA repair in chromatin. We focus on the role of histone post-translational modifications (PTMs) in repair, as well as the molecular mechanisms by which histone mutants affect cellular sensitivity to DNA damage agents and repair activity in chromatin. Importantly, these mechanisms are thought to significantly impact somatic mutation rates in human cancers and potentially contribute to carcinogenesis and other human diseases. For example, a number of the histone mutants studied primarily in yeast have been identified as candidate oncohistone mutations in different cancers. This review highlights these connections and discusses the potential importance of DNA repair in chromatin to human health. [ABSTRACT FROM AUTHOR]

Increasing antimicrobial drug resistance represents a global existential threat. Infection is a particular problem in immunocompromised individuals, such as patients undergoing cancer chemotherapy, due to the targeting of rapidly dividing cells by antineoplastic agents. We recently developed a strategy that targets bacterial nucleotide excision DNA repair (NER) to identify compounds that act as antimicrobial sensitizers specific for patients undergoing cancer chemotherapy. Building on this, we performed a virtual drug screening of a ~120,000 compound library against the key NER protein UvrA. From this, numerous target compounds were identified and of those a candidate compound, Bemcentinib (R428), showed a strong affinity toward UvrA. This NER protein possesses four ATPase sites in its dimeric state, and we found that Bemcentinib could inhibit UvrA's ATPase activity by ~90% and also impair its ability to bind DNA. As a result, Bemcentinib strongly diminishes NER's ability to repair DNA in vitro. To provide a measure of in vivo activity we discovered that the growth of Escherichia coli MG1655 was significantly inhibited when Bemcentinib was combined with the DNA damaging agent 4‐NQO, which is analogous to UV. Using the clinically relevant DNA‐damaging antineoplastic cisplatin in combination with Bemcentinib against the urological sepsis‐causing E. coli strain EC958 caused complete growth inhibition. This study offers a novel approach for the potential development of new compounds for use as adjuvants in antineoplastic therapy. [ABSTRACT FROM AUTHOR]

BackgroundTrichothiodystrophy-1 (TTD1) is an autosomal-recessive disease and caused by mutations in ERCC2, a gene coding for a subunit of the TFIIH transcription and nucleotide-excision repair (NER) factor. In almost half of these patients infectious susceptibility has been reported but the underlying molecular mechanism leading to immunodeficiency is largely unknown.ObjectiveThe aim of this study was to perform extended molecular and immunological phenotyping in patients suffering from TTD1.MethodsCellular immune phenotype was investigated using multicolor flow cytometry. DNA repair efficiency was evaluated in UV-irradiation assays. Furthermore, early BCR activation events and proliferation of TTD1 lymphocytes following DNA damage induction was tested. In addition, we performed differential gene expression analysis in peripheral lymphocytes of TTD1 patients.ResultsWe investigated three unrelated TTD1 patients who presented with recurrent infections early in life of whom two harbored novel ERCC2 mutations and the third patient is a carrier of previously described pathogenic ERCC2 mutations. Hypogammaglobulinemia and decreased antibody responses following vaccination were found. TTD1 B-cells showed accumulation of γ-H2AX levels, decreased proliferation activity and reduced cell viability following UV-irradiation. mRNA sequencing analysis revealed significantly downregulated genes needed for B-cell development and activation. Analysis of B-cell subpopulations showed low numbers of naïve and transitional B-cells in TTD1 patients, indicating abnormal B-cell differentiation in vivo.ConclusionIn summary, our analyses confirmed the pathogenicity of novel ERCC2 mutations and show that ERCC2 deficiency is associated with antibody deficiency most likely due to altered B-cell differentiation resulting from impaired BCR-mediated B-cell activation and activation-induced gene transcription.

The xeroderma pigmentosum protein A (XPA) and replication protein A (RPA) proteins fulfill essential roles in the assembly of the preincision complex in the nucleotide excision repair (NER) pathway. We have previously characterized the two interaction sites, one between the XPA N-terminal (XPA-N) disordered domain and the RPA32 C-terminal domain (RPA32C), and the other with the XPA DNA binding domain (DBD) and the RPA70AB DBDs. Here, we show that XPA mutations that inhibit the physical interaction in either site reduce NER activity in biochemical and cellular systems. Combining mutations in the two sites leads to an additive inhibition of NER, implying that they fulfill distinct roles. Our data suggest a model in which the interaction between XPA-N and RPA32C is important for the initial association of XPA with NER complexes, while the interaction between XPA DBD and RPA70AB is needed for structural organization of the complex to license the dual incision reaction. Integrative structural models of complexes of XPA and RPA bound to single-stranded/double-stranded DNA (ss/dsDNA) junction substrates that mimic the NER bubble reveal key features of the architecture of XPA and RPA in the preincision complex. Most critical among these is that the shape of the NER bubble is far from colinear as depicted in current models, but rather the two strands of unwound DNA must assume a U-shape with the two ss/dsDNA junctions localized in close proximity. Our data suggest that the interaction between XPA and RPA70 is key for the organization of the NER preincision complex.

Maintaining genomic stability and properly repairing damaged DNA is essential to staying healthy and preserving cellular homeostasis. The five major pathways involved in repairing eukaryotic DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end joining (NHEJ), and homologous recombination (HR). When these pathways do not properly repair damaged DNA, genomic stability is compromised and can contribute to diseases such as cancer. It is essential that the causes of DNA damage and the consequent repair pathways are fully understood, yet the initial recruitment and regulation of DNA damage response proteins remains unclear. In this review, the causes of DNA damage, the various mechanisms of DNA damage repair, and the current research regarding the early steps of each major pathway were investigated. [ABSTRACT FROM AUTHOR]

Purpose: The transcription factor NF-E2-related factor 2 (NRF2) is a master regulator widely involved in essential cellular functions such as DNA repair. By clarifying the upstream and downstream links of NRF2 to DNA damage repair, we hope that attention will be drawn to the utilization of NRF2 as a target for cancer therapy. Methods: Query and summarize relevant literature on the role of NRF2 in direct repair, BER, NER, MMR, HR, and NHEJ in pubmed. Make pictures of Roles of NRF2 in DNA Damage Repair and tables of antioxidant response elements (AREs) of DNA repair genes. Analyze the mutation frequency of NFE2L2 in different types of cancer using cBioPortal online tools. By using TCGA, GTEx and GO databases, analyze the correlation between NFE2L2 mutations and DNA repair systems as well as the degree of changes in DNA repair systems as malignant tumors progress. Results: NRF2 plays roles in maintaining the integrity of the genome by repairing DNA damage, regulating the cell cycle, and acting as an antioxidant. And, it possibly plays roles in double stranded break (DSB) pathway selection following ionizing radiation (IR) damage. Whether pathways such as RNA modification, ncRNA, and protein post-translational modification affect the regulation of NRF2 on DNA repair is still to be determined. The overall mutation frequency of the NFE2L2 gene in esophageal carcinoma, lung cancer, and penile cancer is the highest. Genes (50 of 58) that are negatively correlated with clinical staging are positively correlated with NFE2L2 mutations or NFE2L2 expression levels. Conclusion: NRF2 participates in a variety of DNA repair pathways and plays important roles in maintaining genome stability. NRF2 is a potential target for cancer treatment. [ABSTRACT FROM AUTHOR]

Xeroderma pigmentosum (XP) results from biallelic mutations in any of eight genes involved in DNA repair systems, thus defining eight different genotypes (XPA, XPB, XPC, XPD, XPE, XPF, XPG and XP variant or XPV). In addition to cutaneous and ophthalmological features, some patients present with XP neurological disease. It is unknown whether the different neurological signs and their progression differ among groups. Therefore, we aim to characterize the XP neurological disease and its evolution in the heterogeneous UK XP cohort. Patients with XP were followed in the UK National XP Service, from 2009 to 2021. Age of onset for different events was recorded. Cerebellar ataxia and additional neurological signs and symptoms were rated with the Scale for the Assessment and Rating of Ataxia (SARA), the Inventory of Non-Ataxia Signs (INAS) and the Activities of Daily Living questionnaire (ADL). Patients' mutations received scores based on their predicted effects. Data from available ancillary tests were collected. Ninety-three XP patients were recruited. Thirty-six (38.7%) reported neurological symptoms, especially in the XPA, XPD and XPG groups, with early-onset and late-onset forms, and typically appearing after cutaneous and ophthalmological symptoms. XPA, XPD and XPG patients showed higher SARA scores compared to XPC, XPE and XPV. SARA total scores significantly increased over time in XPD (0.91 points/year, 95% confidence interval: 0.61, 1.21) and XPA (0.63 points/year, 95% confidence interval: 0.38, 0.89). Hyporeflexia, hypopallesthaesia, upper motor neuron signs, chorea, dystonia, oculomotor signs and cognitive impairment were frequent findings in XPA, XPD and XPG. Cerebellar and global brain atrophy, axonal sensory and sensorimotor neuropathies, and sensorineural hearing loss were common findings in patients. Some XPC, XPE and XPV cases presented with abnormalities on examination and/or ancillary tests, suggesting underlying neurological involvement. More severe mutations were associated with a faster progression in SARA total score in XPA (0.40 points/year per 1-unit increase in severity score) and XPD (0.60 points/year per 1-unit increase), and in ADL total score in XPA (0.35 points/year per 1-unit increase). Symptomatic and asymptomatic forms of neurological disease are frequent in XP patients, and neurological symptoms can be an important cause of disability. Typically, the neurological disease will be preceded by cutaneous and ophthalmological features, and these should be actively searched in patients with idiopathic late-onset neurological syndromes. Scales assessing cerebellar function, especially walking and speech, and disability can show progression in some of the groups. Mutation severity can be used as a prognostic biomarker for stratification purposes in clinical trials. [ABSTRACT FROM AUTHOR]

In all cells—from bacteria to humans—nucleotide excision repair (NER) is a highly conserved, versatile DNA repair pathway that is responsible for the removal of a wide variety of DNA helix-distorting lesions arising from both endogenous and exogenous sources. In many organisms including bacteria, fungi, animals, and plants, NER occurs through two sub-pathways: the global genome NER (GG-NER) pathway and the transcription-coupled NER (TC-NER) pathway. Although essential factors and basic steps involved in NER have been identified, mechanisms and stages of their assembly process are not well understood. In this review, we summarize recent literature about protein interaction networks that manifest during initial stages of bacterial NER pathway while highlighting some of the key functional studies. [ABSTRACT FROM AUTHOR]

DNA helicase activity is essential for the vital DNA metabolic processes of recombination, replication, transcription, translation, and repair. Recently, an unexpected, rapid exponential ATP‐stimulated DNA unwinding rate was observed from an Archaeoglobus fulgidus helicase (AfXPB) as compared to the slower conventional helicases from Sulfolobus tokodaii, StXPB1 and StXPB2. This unusual rapid activity suggests a "molecular wrench" mechanism arising from the torque applied by AfXPB on the duplex structure in transitioning from open to closed conformations. However, much remains to be understood. Here, we investigate the concentration dependence of DNA helicase binding and ATP‐stimulated kinetics of StXPB2 and AfXPB, as well as their binding and activity in Bax1 complexes, via an electrochemical assay with redox‐active DNA monolayers. StXPB2 ATP‐stimulated activity is concentration‐independent from 8 to 200 nM. Unexpectedly, AfXPB activity is concentration‐dependent in this range, with exponential rate constants varying from seconds at concentrations greater than 20 nM to thousands of seconds at lower concentrations. At 20 nM, rapid exponential signal decay ensues, linearly reverses, and resumes with a slower exponential decay. This change in AfXPB activity as a function of its concentration is rationalized as the crossover between the fast molecular wrench and slower conventional helicase modes. AfXPB‐Bax1 inhibits rapid activity, whereas the StXPB2‐Bax1 complex induces rapid kinetics at higher concentrations. This activity is rationalized with the crystal structures of these complexes. These findings illuminate the different physical models governing molecular wrench activity for improved biological insight into a key factor in DNA repair. [ABSTRACT FROM AUTHOR]

Background: Thyroid hormone signaling is essential for development, metabolism, and response to stress but declines during aging, the cause of which is unknown. DNA damage accumulating with time is a main cause of aging, driving many age-related diseases. Previous studies in normal and premature aging mice, due to defective DNA repair, indicated reduced hepatic thyroid hormone signaling accompanied by decreased type 1 deiodinase (DIO1) and increased DIO3 activities. We investigated whether aging-related changes in deiodinase activity are driven by systemic signals or represent cell- or organ-autonomous changes. Methods: We quantified liver and plasma thyroid hormone concentrations, deiodinase activities and expression of T3-responsive genes in mice with a global, liver-specific and for comparison brain-specific inactivation of Xpg, one of the endonucleases critically involved in multiple DNA repair pathways. Results: Both in global and liver-specific Xpg knockout mice, hepatic DIO1 activity was decreased. Interestingly, hepatic DIO3 activity was increased in global, but not in liver-specific Xpg mutants. Selective Xpg deficiency and premature aging in the brain did not affect liver or systemic thyroid signaling. Concomitant with DIO 1 inhibition, Xpg−/− and Alb-Xpg mice displayed reduced thyroid hormone-related gene expression changes, correlating with markers of liver damage and cellular senescence. Conclusions: Our findings suggest that DIO1 activity during aging is predominantly modified in a tissue-autonomous manner driven by organ/cell-intrinsic accumulating DNA damage. The increase in hepatic DIO3 activity during aging largely depends on systemic signals, possibly reflecting the presence of circulating cells rather than activity in hepatocytes.

Pierisin is a DNA-targeting ADP-ribosyltransferase found in cabbage white butterfly (Pieris rapae). Pierisin transfers an ADP-ribosyl moiety to the 2-amino group of the guanine residue in DNA, yielding N2-(ADP-ribos-1-yl)-2′-deoxyguanosine (N2-ADPR-dG). Generally, such chemically modified DNA is recognized as DNA damage and elicits cellular responses, including DNA repair pathways. In Escherichia coli and human cells, it has been experimentally demonstrated that N2-ADPR-dG is a substrate of the nucleotide excision repair system. Although DNA repair machineries can remove most lesions, some unrepaired damages frequently lead to mutagenesis through DNA replication. Replication past the damaged DNA template is called translesion DNA synthesis (TLS). In vitro primer extension experiments have shown that eukaryotic DNA polymerase κ is involved in TLS across N2-ADPR-dG. In many cases, TLS is error-prone and thus a mutagenic process. Indeed, the induction of G:C to T:A and G:C to C:G mutations by N2-ADPR-dG in the hypoxanthine phosphoribosyltransferase gene mutation assay with Chinese hamster cells and supF shuttle vector plasmids assay using human fibroblasts has been reported. This review provides a detailed overview of DNA repair, TLS and mutagenesis of N2-ADPR-dG induced by cabbage butterfly pierisin-1.

Nucleotide excision repair (NER) is the most universal repair pathway, which removes a wide range of DNA helix-distorting lesions caused by chemical or physical agents. The final steps of this repair process are gap-filling repair synthesis and subsequent ligation. XPA is the central NER scaffolding protein factor and can be involved in post-incision NER stages. Replication machinery is loaded after the first incision of the damaged strand that is performed by the XPF–ERCC1 nuclease forming a damaged 5′-flap processed by the XPG endonuclease. Flap endonuclease I (FEN1) is a critical component of replication machinery and is absolutely indispensable for the maturation of newly synthesized strands. FEN1 also contributes to the long-patch pathway of base excision repair. Here, we use a set of DNA substrates containing a fluorescently labeled 5′-flap and different size gap to analyze possible repair factor–replication factor interactions. Ternary XPA–FEN1–DNA complexes with each tested DNA are detected. Furthermore, we demonstrate XPA–FEN1 complex formation in the absence of DNA due to protein–protein interaction. Functional assays reveal that XPA moderately inhibits FEN1 catalytic activity. Using fluorescently labeled XPA, formation of ternary RPA–XPA–FEN1 complex, where XPA accommodates FEN1 and RPA contacts simultaneously, can be proposed. We discuss possible functional roles of the XPA–FEN1 interaction in NER related DNA resynthesis and/or other DNA metabolic processes where XPA can be involved in the complex with FEN1.

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. [ABSTRACT FROM AUTHOR]

RNA polymerase II moves along genes to decode genetic information stored in the mammalian genome into messenger RNA and different forms of non‐coding RNA. However, the transcription process is frequently challenged by DNA lesions caused by exogenous and endogenous insults, among which helix‐distorting DNA lesions and double‐stranded DNA breaks are particularly harmful for cell survival. In response to such DNA damage, RNA polymerase II transcription is regulated both locally and globally by multi‐layer mechanisms, whereas transcription‐blocking lesions are repaired before transcription can recover. Failure in DNA damage repair will cause genome instability and cell death. Although recent studies have expanded our understanding of RNA polymerase II regulation confronting DNA lesions, it is still not always clear what the direct contribution of RNA polymerase II is in the DNA damage repair processes. In this review, we focus on how RNA polymerase II and transcription are both repressed by transcription stalling lesions such as DNA‐adducts and double strand breaks, as well as how they are actively regulated to support the cellular response to DNA damage and favour the repair of lesions. [ABSTRACT FROM AUTHOR]

Strain Deinococcus irradiatisoli 17bor-2 was isolated from a soil sample exposed to γ radiation at Seoul Women's University, Republic of Korea. The genus Deinococcus is a Gram-negative, coccus-shaped, and extremophilic bacterium, well renowned as being a radiation-resistant bacterium. Therefore, the mechanism behind the resistance to radiation and the gene responsible for the resistance could be helpful for detailed experimental studies with biotechnological applications. To study the involvement of genes in UV radiation resistance in strain 17bor-2, the genomic DNA of the strain was sequenced and constructed using the Pacific Biosciences RS II system. In addition, the complete genome sequence of strain 17bor-2 was annotated and interpreted using the Genomes–Expert Review (IMG-ER) system, along with Prodigal and JGI GenePRIMP analysis. The genome analysis of strain 17bor-2 revealed evidence of excinuclease UvrABC genes, which are key enzymes in the nucleotide excision repair (NER) mechanism, as well as genes from the recA-dependent and recQ pathways. The genome of strain Deinococcus irradiatisoli 17bor-2 was a circular chromosome comprising 3,052,043 bp with a GC content of 67.0%, including 2911 coding sequences (CDs), 49 tRNA genes, and 9 rRNA genes. In addition, their complete genome sequence annotation features provided evidence that radiation resistance genes play a central part in adaptation against extreme environmental conditions. In recent decades, excision repair genes have been indicated in considerable detail for both prokaryote and eukaryote resistance against UV-C radiation. [ABSTRACT FROM AUTHOR]

Abstract Background Endometrial cancer (EC) arises from uterine endometrium tissue and is the most prevalent cancer of the female reproductive tract in developed countries. It has been predicted that the global prevalence of EC will increase in part because of its positive association with economic growth and lifestyle. The majority of EC presented with endometrioid histology and mutations in the tumor suppressor gene PTEN, resulting in its loss of function. PTEN negatively regulates the PI3K/Akt/mTOR axis of cell proliferation and thus serves as a tumorigenesis gatekeeper. Through its chromatin functions, PTEN is also implicated in genome maintenance procedures. However, our comprehension of how DNA repair occurs in the absence of PTEN function in EC is inadequate. Methods We utilized The Cancer Genome Atlas (TCGA) data analysis to establish a correlation between PTEN and DNA damage response genes in EC, followed by a series of cellular and biochemical assays to elucidate a molecular mechanism utilizing the AN3CA cell line model for EC. Results The TCGA analyses demonstrated an inverse correlation between the expression of the damage sensor protein of nucleotide excision repair (NER), DDB2, and PTEN in EC. The transcriptional activation of DDB2 is mediated by the recruitment of active RNA polymerase II to the DDB2 promoter in the PTEN-null EC cells, revealing a correlation between increased DDB2 expression and augmented NER activity in the absence of PTEN. Conclusion Our study indicated a causal relationship between NER and EC that may be exploited in disease management.

All tumors have DNA mutations, and a predictive understanding of those mutations could inform clinical treatments. However, 40% of the mutations are variants of unknown significance (VUS), with the challenge being to objectively predict whether a VUS is pathogenic and supports the tumor or whether it is benign. To objectively decode VUS, we mapped cancer sequence data and evolutionary trace (ET) scores onto crystallography and cryo-electron microscopy structures with variant impacts quantitated by evolutionary action (EA) measures. As tumors depend on helicases and nucleases to deal with transcription/replication stress, we targeted helicase-nuclease-RPA complexes: (1) XPB-XPD (within TFIIH), XPF-ERCC1, XPG, and RPA for transcription and nucleotide excision repair pathways and (2) BLM, EXO5, and RPA plus DNA2 for stalled replication fork restart. As validation, EA scoring predicts severe effects for most disease mutations, but disease mutants with low ET scores not only are likely destabilizing but also disrupt sophisticated allosteric mechanisms. For sites of disease mutations and VUS predicted to be severe, we found strong co-localization to ordered regions. Rare discrepancies highlighted the different survival requirements between disease and tumor mutations, as well as the value of examining proteins within complexes. In a genome-wide analysis of 33 cancer types, we found correlation between the number of mutations in each tumor and which pathways or functional processes in which the mutations occur, revealing different mutagenic routes to tumorigenesis. We also found upregulation of ancient genes including BLM, which supports a non-random and concerted cancer process: reversion to a unicellular, proliferation-uncontrolled, status by breaking multicellular constraints on cell division. Together, these genes and global analyses challenge the binary "driver" and "passenger" mutation paradigm, support a gradient impact as revealed by EA scoring from moderate to severe at a single gene level, and indicate reduced regulation as well as activity. The objective quantitative assessment of VUS scoring and gene overexpression in the context of functional interactions and pathways provides insights for biology, oncology, and precision medicine.

Critical for transcription initiation and bulky lesion DNA repair, TFIIH provides an exemplary system to connect molecular mechanisms to biological outcomes due to its strong genetic links to different specific human diseases. Recent advances in structural and computational biology provide a unique opportunity to re-examine biologically relevant molecular structures and develop possible mechanistic insights for the large dynamic TFIIH complex. TFIIH presents many puzzles involving how its two SF2 helicase family enzymes, XPB and XPD, function in transcription initiation and repair: how do they initiate transcription, detect and verify DNA damage, select the damaged strand for incision, coordinate repair with transcription and cell cycle through Cdk-activating-kinase (CAK) signaling, and result in very different specific human diseases associated with cancer, aging, and development from single missense mutations? By joining analyses of breakthrough cryo-electron microscopy (cryo-EM) structures and advanced computation with data from biochemistry and human genetics, we develop unified concepts and molecular level understanding for TFIIH functions with a focus on structural mechanisms. We provocatively consider that TFIIH may have first evolved from evolutionary pressure for TCR to resolve arrested transcription blocks to DNA replication and later added its key roles in transcription initiation and global DNA repair. We anticipate that this level of mechanistic information will have significant impact on thinking about TFIIH, laying a robust foundation suitable to develop new paradigms for DNA transcription initiation and repair along with insights into disease prevention, susceptibility, diagnosis and interventions.

In plants, prolonged exposure to ultraviolet (UV) radiation causes harmful DNA lesions. Nucleotide excision repair (NER) is an important DNA repair mechanism that operates via two pathways: transcription coupled repair (TC-NER) and global genomic repair (GG-NER). In plants and mammals, TC-NER is initiated by the Cockayne Syndrome A and B (CSA/CSB) complex, whereas GG-NER is initiated by the Damaged DNA Binding protein 1/2 (DDB1/2) complex. In the yeast Saccharomyces cerevisiae (S. cerevisiae), GG-NER is initiated by the Radiation Sensitive 7 and 16, (RAD7/16) complex. Arabidopsis thaliana has two homologues of yeast RAD16, At1g05120 and At1g02670, which we named AtRAD16 and AtRAD16b, respectively. In this study, we characterized the roles of AtRAD16 and AtRAD16b. Arabidopsis rad16 and rad16b null mutants exhibited increased UV sensitivity. Moreover, AtRAD16 overexpression increased plant UV tolerance. Thus, AtRAD16 and AtRAD16b contribute to plant UV tolerance and growth. Additionally, we found physical interaction between AtRAD16 and AtRAD7. Thus, the Arabidopsis RAD7/16 complex is functional in plant NER. Furthermore, AtRAD16 makes a significant contribution to Arabidopsis UV tolerance compared to the DDB1/2 and the CSB pathways. This is the first time the role and interaction of DDB1/2, RAD7/16, and CSA/CSB components in a single system have been studied. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Nucleotide excision repair (NER) has been associated with various types of malignant tumors. However, the precise roles of nucleotide excision repair-related genes (NERGs) in acute myeloid leukemia (AML) remain incompletely understood. Hence, this study aimed to develop a prognostic signature incorporating NERGs in AML, which could potentially predict patient outcomes. MATERIALS AND METHODS: By querying the Genotype-Tissue Expression (GTEx), The Cancer Genome Atlas (TCGA), and Gene Expression Omnibus (GEO) databases, we acquired RNA-seq data and clinical information pertaining to AML. To identify differentially expressed NERGs (DE-NERGs), we employed the Wilcoxon rank-sum test. Based on the expression patterns of DE-NERGs with prognostic significance, patients were categorized into two subgroups. A prognostic signature was developed through univariate Cox regression and least absolute shrinkage and selection operator (LASSO) analyses to compare the differentially expressed genes (DEGs) between these two groups. Additionally, a nomogram was constructed using multivariate analysis. The biological pathways involved were elucidated through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, gene set variation analysis (GSVA), and gene set enrichment analysis (GSEA). RESULTS: We developed a prognostic model based on an 11-gene signature. Furthermore, the risk score derived from this model was demonstrated to independently serve as a prognostic marker for patients diagnosed with AML. CONCLUSIONS: Our prognostic model, based on NERGs, was developed and validated to provide insights into the onset and progression of AML and establish a foundation for more effective treatment. Our findings not only contribute to clinical decision-making but also underscore the significance of nucleotide excision repair. Furthermore, they may pave the way for the development of targeted therapeutic strategies specifically focused on this process. [ABSTRACT FROM AUTHOR]

Ultraviolet (UV) light primarily causes C > T substitutions in lesion-forming dipyrimidine sequences. However, many of the key driver mutations in melanoma do not fit this canonical UV signature, but are instead caused by T > A, T > C, or C > A substitutions. To what extent exposure to the UVB or UVA spectrum of sunlight can induce these noncanonical mutation classes, and the molecular mechanism involved is unclear. Here, we repeatedly exposed wild-type or repair-deficient yeast (Saccharomyces cerevisiae) to UVB or UVA light and characterized the resulting mutations by whole genome sequencing. Our data indicate that UVB induces C > T and T > C substitutions in dipyrimidines, and T > A substitutions that are often associated with thymine–adenine (TA) sequences. All of these mutation classes are induced in nucleotide excision repair–deficient cells and show transcriptional strand asymmetry, suggesting they are caused by helix-distorting UV photoproducts. In contrast, UVA exposure induces orders of magnitude fewer mutations with a distinct mutation spectrum. UVA-induced mutations are elevated in Ogg1-deficient cells, and the resulting spectrum consists almost entirely of C > A/G > T mutations, indicating they are likely derived from oxidative guanine lesions. These mutations show replication asymmetry, with elevated G > T mutations on the leading strand, suggesting there is a strand bias in the removal or bypass of guanine lesions during replication. Finally, we develop a mutation reporter to show that UVA induces a G > T reversion mutation in yeast that mimics the oncogenic NRAS Q61K mutation in melanoma. Taken together, these findings indicate that UVA and UVB exposure can induce many of the noncanonical mutation classes that cause driver mutations in melanoma. [ABSTRACT FROM AUTHOR]

Cigarette smoking (CS) is a major cause of various serious diseases due to tobacco chemicals. There is evidence suggesting that CS has been linked with the DNA damage repair system, as it can affect genomic stability, inducing genetic changes in the genes involved in the repair system, specifically the nucleotide excision repair (NER) pathway, affecting the function and/or regulation of these genes. Single nucleotide polymorphism (SNP), along with CS, can affect the work of the NER pathway and, therefore, could lead to different diseases. This study explored the association of four SNPs in both XPA and XPC genes with CS in the Saudi population. The Taq Man genotyping assay was used for 220 healthy non-smokers (control) and 201 healthy smokers to evaluate four SNPs in the XPA gene named rs10817938, rs1800975, rs3176751, and rs3176752 and four SNPs in the XPC gene called rs1870134, rs2228000, rs2228001, and rs2607775. In the XPA gene, SNP rs3176751 showed a high-risk association with CS-induced diseases with all clinical parameters, including CS duration, CS intensity, gender, and age of smokers. On the other hand, SNP rs1800975 showed a statistically significant low-risk association with all clinical parameters. In addition, rs10817938 showed a high-risk association only with long-term smokers and a low-risk association only with younger smokers. A low-risk association was found in SNP rs3176752 with older smokers. In the XPC gene, SNP rs2228001 showed a low-risk association only with female smokers. SNP rs2607775 revealed a statistically significant low-risk association with CS-induced diseases, concerning all parameters, except for male smokers. However, SNP rs2228000 and rs1870134 showed no association with CS. Overall, the study results demonstrated possible significant associations (effector/and protector) between CS and SNPs polymorphisms in DNA repair genes, such as XPA and XPC, except for rs2228000 and rs1870134 polymorphisms. [ABSTRACT FROM AUTHOR]

Transcription-coupled repair (TCR), discovered as preferential nucleotide excision repair of UV-induced cyclobutane pyrimidine dimers located in transcribed mammalian genes compared to those in nontranscribed regions of the genome, is defined as faster repair of the transcribed strand versus the nontranscribed strand in transcribed genes. The phenomenon, universal in model organisms including Escherichia coli, yeast, Arabidopsis, mice, and humans, involves a translocase that interacts with both RNA polymerase stalled at damage in the transcribed strand and nucleotide excision repair proteins to accelerate repair. Drosophila, a notable exception, exhibits TCR but lacks an obvious TCR translocase. Mutations inactivating TCR genes cause increased damage-induced mutagenesis in E. coli and severe neurological and UV sensitivity syndromes in humans. To date, only E. coli TCR has been reconstituted in vitro with purified proteins. Detailed investigations of TCR using genome-wide next-generation sequencing methods, cryo–electron microscopy, single-molecule analysis, and other approaches have revealed fascinating mechanisms. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) is a cellular process that removes damaged bases arising from exogenous and endogenous sources including reactive oxygen species, alkylation agents, and ionizing radiation. BER is mediated by the actions of multiple proteins which work in a highly concerted manner to resolve DNA damage efficiently to prevent toxic repair intermediates. During the initiation of BER, the damaged base is removed by one of 11 mammalian DNA glycosylases, resulting in abasic sites. Many DNA glycosylases are product-inhibited by binding to the abasic site more avidly than the damaged base. Traditionally, apurinic/apyrimidinic endonuclease 1, APE1, was believed to help turn over the glycosylases to undergo multiple rounds of damaged base removal. However, in a series of papers from our laboratory, we have demonstrated that UV-damaged DNA binding protein (UV-DDB) stimulates the glycosylase activities of human 8-oxoguanine glycosylase (OGG1), MUTY DNA glycosylase (MUTYH), alkyladenine glycosylase/N-methylpurine DNA glycosylase (AAG/MPG), and single-strand selective monofunctional glycosylase (SMUG1), between three- and five-fold. Moreover, we have shown that UV-DDB can assist chromatin decompaction, facilitating access of OGG1 to 8-oxoguanine damage in telomeres. This review summarizes the biochemistry, single-molecule, and cell biology approaches that our group used to directly demonstrate the essential role of UV-DDB in BER. [ABSTRACT FROM AUTHOR]

Periodontitis is a chronic inflammatory disease that affects the supporting structures of teeth. In the literature, the association between the pathogenicity of bacteria and environmental factors in this regard have been extensively examined. In the present study, we will shed light on the potential role that epigenetic change can play on different facets of its process, more particularly the modifications concerning the genes involved in inflammation, defense, and immune systems. Since the 1960s, the role of genetic variants in the onset and severity of periodontal disease has been widely demonstrated. These make some people more susceptible to developing it than others. It has been documented that the wide variation in its frequency for various racial and ethnic populations is due primarily to the complex interplay among genetic factors with those affecting the environment and the demography. In molecular biology, epigenetic modifications are defined as any change in the promoter for the CpG islands, in the structure of the histone protein, as well as post-translational regulation by microRNAs (miRNAs), being known to contribute to the alteration in gene expression for complex multifactorial diseases such as periodontitis. The key role of epigenetic modification is to understand the mechanism involved in the gene-environment interaction, and the development of periodontitis is now the subject of more and more studies that attempt to identify which factors are stimulating it, but also affect the reduced response to therapy. [ABSTRACT FROM AUTHOR]

Fish at early life stages are sensitive to temperature change because of their narrower temperature tolerance ranges. Initiated by damage detection, DNA mismatch repair (MMR) and nucleotide excision repair (NER) maintain genome integrity respectively by eliminating mismatched nucleotides and helix-distorting DNA lesions. As discharge of heated effluent from power plants may elevate water temperatures to only 2 to 6 °C higher than ambient, this study explored if temperatures within this range affected MMR and NER-linked damage detection activities in fish embryos using zebrafish (Danio rerio) embryo as a model organism. Exposure of early embryos at 10 h post fertilization (hpf) to a warmer temperature at + 4.5 °C for 30 min enhanced damage recognition activities targeting UV-induced cyclobutane pyrimidine dimers (CPDs) and (6–4) photoproducts (6-4PPs) that distorted helical structures. Conversely, photolesions sensing activities were inhibited in 24 hpf mid-early embryos under the same stress conditions. A much higher temperature at + 8.5 °C imposed similar effects on UV damage detection. A mild heat stress at + 2.5 °C for 30 min, however, repressed both CPD and 6-4PP binding activities in 10 and 24 hpf embryos. Inhibition of damage recognition under mild heat stress impeded the overall NER capacity evidenced by a transcription-based repair assay. Warmer water temperatures at + 2.5 and + 4.5 °C also inhibited G-T mismatch binding activities in 10 and 24 hpf embryos, but G-T recognition was more sensitive to + 4.5 °C stress. Inhibition of G-T binding partially correlated with a downregulation of Sp1 transcription factor activity. Our results showed the potential of water temperature elevation within 2 to 4.5 °C to disturb DNA damage repair in fish at embryonic stages. [ABSTRACT FROM AUTHOR]

Simple Summary: Xeroderma pigmentosum (XP) is a genetic disease caused by DNA repair deficiency. We diagnosed 181 XP patients from 1982 to 2022 in France. The patients are very sensitive to sunlight and will rapidly develop skin cancers in exposed body sites. However, thanks to better protection, XP patients live much longer than in the past. This better survival is associated, for the French XP patients originating from North Africa and bearing the same founder mutation in the XPC gene, with a very high risk to develop aggressive and lethal internal cancers, particularly hematological malignancies, and brain, gynecological, and thyroid tumors. This is a new threat hanging over all XP-C patients. The molecular origins of internal tumors in this particular set of DNA repair-deficient patients are discussed. It is important that physicians and XP families be aware of this high risk to prevent and look for early diagnosis of internal tumor development in XP patients. Background: Xeroderma pigmentosum (XP) is a rare genetic disorder characterized by a high incidence of skin cancers. These patients are deficient in nucleotide excision repair caused by mutations in one of the 7 XP genes. Methods: We diagnosed 181 XP patients using UV-induced DNA repair measurements and/or DNA sequencing from 1982 to 2022 in France. Results: As all XP patients, the French ones are very sensitive to UV exposure but since they are usually very well protected, they develop relatively few skin cancers. A majority of French XP patients originate from North Africa and bear a founder mutation on the XPC gene. The striking discovery is that these patients are at a very high risk to develop aggressive and lethal internal tumors such as hematological malignancies (more than a 100-fold risk compared to the general population for myelodysplasia/leukemia) with a median age of death of 25 years, and brain, gynecological, and thyroid tumors with even lower median ages of death. The high mutation rates found in XP-C internal tumors allow us to think that these XP patients could be successfully treated by immunotherapies. A full analysis of the molecular origins of these DNA repair-deficient tumors is discussed. Several explanations for this high predisposition risk are proposed. Conclusions: As the age of the XP population is increasing due to better photo-protection, the risk of lethal internal tumors is a new Damocles sword that hangs over XP-C patients. This review of the French cohort is of particular importance for alerting physicians and families to the prevention and early detection of aggressive internal tumors in XP patients. [ABSTRACT FROM AUTHOR]

Genomes are constantly in flux, undergoing changes due to recombination, repair and mutagenesis. In vivo, many of such changes are studies using reporters for specific types of changes, or through cytological studies that detect changes at the single-cell level. Single molecule assays, which are reviewed here, can detect transient intermediates and dynamics of events. Biochemical assays allow detailed investigation of the DNA and protein activities of each step in a repair, recombination or mutagenesis event. Each type of assay is a powerful tool but each comes with its particular advantages and limitations. Here the most commonly used assays are reviewed, discussed, and presented as the guidelines for future studies.

Abstract Background Xeroderma pigmentosum is an extremely serious genetic disorder defined by sensitivity to sunlight, resulting in sunburn and pigment changes. If patients are not completely protected from ultraviolet radiation, xeroderma pigmentosum is characterized by a greatly increased risk of sunlight-induced cutaneous neoplasms. There is no standard therapy for skin cancer of xeroderma pigmentosum. However, immune checkpoint inhibitors were reported to increase response rates and improve outcomes and life expectancy in patients with various cancers, including squamous cell carcinoma in xeroderma pigmentosum. In this paper, we report on a patient with xeroderma pigmentosum from a consanguineous family with recurrent facial chemotherapy-resistant squamous cell carcinoma lesions treated successfully with an anti-programmed cell death protein 1 monoclonal antibody in both relapses. Case presentation A 7-year-old Turkish male was referred to our oncology department for recurring squamous cell carcinoma after local excision of the tumor over his nose. The lesion was a rapidly growing lesion, measuring 8 × 4 cm in size. Physical examination revealed that he also had hemorrhagic crusted plaques and nodules over both eyelids and upper lip, with multiple hypo- and hyperpigmented punctate lesions all over his body. After two more cycles of chemotherapy, progressive disease was noted, and a new lesion on the right eyelid caused blurred vision. Anti-programmed cell death protein 1 antibody treatment was planned with concomitant radiotherapy. He received nivolumab every 3 weeks for 4 months, improving his vision. No new lesions or active complaints have been observed in the current situation, and complete remission has been achieved. On the last admission, the patient was clinically diagnosed with xeroderma pigmentosum. Owing to the condition’s genetic heterogeneity, whole-exome sequencing was performed with Ion Proton next-generation sequencing platform, and the c.2250 + 1G>A splice site mutation of the XPC gene was detected in the homozygous state. Conclusions The clinical report emphasizes the importance of clinical awareness and crucial early diagnosis of xeroderma pigmentosum and presents a novel causative homozygous c.2250 + 1G>A splice site mutation. Our case proves that next-generation sequencing is an effective method for the rapid diagnosis and determination of xeroderma pigmentosum genetic etiology.

The Rad4-Rad23-Rad33 complex plays an essential anti-ultraviolet (UV) role depending on nucleotide excision repair (NER) in budding yeast but has been rarely studied in filamentous fungi, which possess two Rad4 paralogs (Rad4A/B) and orthologous Rad23 and rely on the photorepair of UV-induced DNA lesions, a distinct mechanism behind the photoreactivation of UV-impaired cells. Previously, nucleocytoplasmic shuttling Rad23 proved to be highly efficient in the photoreactivation of conidia inactivated by UVB, a major component of solar UV, due to its interaction with Phr2 in Beauveria bassiana, a wide-spectrum insect mycopathogen lacking Rad33. Here, either Rad4A or Rad4B was proven to localize exclusively in the nucleus and interact with Rad23, which was previously shown to interact with the white collar protein WC2 as a regulator of two photorepair-required photolyases (Phr1 and Phr2) in B. bassiana. The Δrad4A mutant lost ~80% of conidial UVB resistance and ~50% of activity in the photoreactivation of UVB-inactivated conidia by 5 h light exposure. Intriguingly, the reactivation rates of UVB-impaired conidia were observable only in the presence of rad4A after dark incubation exceeding 24 h, implicating extant, but infeasible, NER activity for Rad4A in the field where night (dark) time is too short. Aside from its strong anti-UVB role, Rad4A played no other role in B. bassiana's lifecycle while Rad4B proved to be functionally redundant. Our findings uncover that the anti-UVB role of Rad4A depends on the photoreactivation activity ascribed to its interaction with Rad23 linked to WC2 and Phr2 and expands a molecular basis underlying filamentous fungal adaptation to solar UV irradiation on the Earth's surface. [ABSTRACT FROM AUTHOR]

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO−), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO− scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs. [ABSTRACT FROM AUTHOR]

Dietary restriction (DR) is a universal anti-aging intervention, which reduces age-related nervous system pathologies and neurological decline. The degree to which the neuroprotective effect of DR operates by attenuating cell intrinsic degradative processes rather than influencing non-cell autonomous factors such as glial and vascular health or systemic inflammatory status is incompletely understood. Following up on our finding that DR has a remarkably large beneficial effect on nervous system pathology in wholebody DNA repair-deficient progeroid mice, we show here that DR also exerts strong neuroprotection in mouse models in which a single neuronal cell type, i.e., cerebellar Purkinje cells, experience genotoxic stress and consequent premature aging-like dysfunction. Purkinje cell specific hypomorphic and knock-out ERCC1 mice on DR retained 40 and 25% more neurons, respectively, with equal protection against P53 activation, and alike results from whole-body ERCC1-deficient mice. Our findings show that DR strongly reduces Purkinje cell death in our Purkinje cell-specific accelerated aging mouse model, indicating that DR protects Purkinje cells from intrinsic DNAdamage-driven neurodegeneration. [ABSTRACT FROM AUTHOR]

Simple Summary: The DNA damage response (DDR) is frequently altered in ovarian cancer (OC), which can be exploited for therapeutic purposes. Moreover, targeting DDR signaling pathways has become an attractive strategy for increasing the effect of DNA-damaging drugs and overcoming chemoresistance. Here, we review the main DDR pathways and their alterations in OC. We also recapitulate the preclinical and clinical studies that target the DDR for the treatment of the disease. The DNA damage response (DDR), a set of signaling pathways for DNA damage detection and repair, maintains genomic stability when cells are exposed to endogenous or exogenous DNA-damaging agents. Alterations in these pathways are strongly associated with cancer development, including ovarian cancer (OC), the most lethal gynecologic malignancy. In OC, failures in the DDR have been related not only to the onset but also to progression and chemoresistance. It is known that approximately half of the most frequent subtype, high-grade serous carcinoma (HGSC), exhibit defects in DNA double-strand break (DSB) repair by homologous recombination (HR), and current evidence indicates that probably all HGSCs harbor a defect in at least one DDR pathway. These defects are not restricted to HGSCs; mutations in ARID1A, which are present in 30% of endometrioid OCs and 50% of clear cell (CC) carcinomas, have also been found to confer deficiencies in DNA repair. Moreover, DDR alterations have been described in a variable percentage of the different OC subtypes. Here, we overview the main DNA repair pathways involved in the maintenance of genome stability and their deregulation in OC. We also recapitulate the preclinical and clinical data supporting the potential of targeting the DDR to fight the disease. [ABSTRACT FROM AUTHOR]

In this study, the co-culture system of Caco-2 and HaCaT cells was established, and the inhibitory effect of punicalagin on cyclobutene pyrimidine dimers (CPD) which was one major type of UVB-induced DNA lesions was evaluated by immunofluorescence. Then the effects of punicalagin on transcription and expression levels of nucleotide excision repair (NER) related genes was further investigated by quantitative real-time PCR, immunoprecipitation and western-blotting. The results showed that punicalagin reduced CPD level in HaCaT cells, upregulated xeroderma pigmentosum group C (XPC) mRNA level and attenuated XPA acetylation in a SIRT1-dependent manner. This showed, punicalagin's efficacy against UVB-induced photodamage in the co-culture system of Caco-2 and HaCaT cells was probably activated by the NER related XPC/ XPA which depended on the deacetylation activity of SIRT1. [ABSTRACT FROM AUTHOR]

Nucleotide excision repair (NER) is a major pathway to deal with bulky adducts induced by various environmental toxins in all cellular organisms. The two sub-pathways of NER, global genome repair (GGR) and transcription-coupled repair (TCR), differ in the damage recognition modes. In this review, we describe the molecular mechanism of NER in mammalian cells, especially the details of damage recognition steps in both sub-pathways. We also introduce new sequencing methods for genome-wide mapping of NER, as well as recent advances about NER in chromatin by these methods. Finally, the roles of NER factors in repairing oxidative damages and resolving R-loops are discussed.

Abstract Background Xeroderma pigmentosum (XP) is a rare, autosomal, recessive DNA repair-deficiency disorder with a frequency of 1–3 per million livebirths in Europe and USA but with higher frequencies in isolated islands or in countries with a high level of consanguinity. XP is characterized by high incidence of skin cancers on sun-exposed sites. Recent improvement in life expectancy of XP patients suggests an increased risk of frequently aggressive and lethal internal tumors. Our purpose was to quantify relative risks of internal tumor development for XP patients by tumor type, XP-subtype, patients’ ages and ethnicity through comparison with the US general population. Methods We analyzed four independent international well-characterized XP cohorts (from USA, UK, France and Brazil) with a total of 434 patients, where 11.3% developed internal tumors and compared them to the American general population. We also compiled, through PubMed/Medline, a dataset of 89 internal tumors in XP patients published between 1958 and 2020. Results In the combined 4-XP cohort, relative risk of internal tumors was 34 (95% confidence interval (CI) 25–47) times higher than in the general population (p-value = 1.0E−47) and tumor arose 50 years earlier. The XP-C group was at the highest risk for the 0–20 years old-patients (OR = 665; 95% CI 368–1200; p-value = 4.3E−30). The highest risks were observed for tumors of central nervous system (OR = 331; 95% CI 171–641; p-value = 2.4E−20), hematological malignancies (OR = 120; 95% CI 77–186; p-value = 3.7E−36), thyroid (OR = 74; 95% CI 31–179; p-value = 1.2E−8) and gynecological tumors (OR = 91; 95% CI 42–193; p-value = 3.5E−12). The type of mutation on the XPC gene is associated with different classes of internal tumors. The majority of French XP-C patients (80%) are originated from North Africa and carried the XPC delTG founder mutation specific from the South Mediterranean area. The OR is extremely high for young (0–20 years) patients with more than 1300-fold increase for the French XPs carrying the founder mutation. Conclusion Because the age of XP population is increasing due to better sun-protection and knowledge of the disease, these results are of particular importance for the physicians to help in early prevention and detection of internal tumors in their XP patients. Few preventive blood analyses or simple medical imaging may help to better detect early cancer appearance in this population.

Nucleotide excision repair (NER) clears genomes of DNA adducts formed by UV light, environmental agents, and antitumor drugs. Gene mutations that lead to defects in the core NER reaction cause the skin cancer-prone disease xeroderma pigmentosum. In NER, DNA lesions are excised within an oligonucleotide of 25–30 residues via a complex, multi-step reaction that is regulated by protein-protein interactions. These interactions were first characterized in the 1990s using pull-down, co-IP and yeast two-hybrid assays. More recently, high-resolution structures and detailed functional studies have started to yield detailed pictures of the progression along the NER reaction coordinate. In this review, we highlight how the study of interactions among proteins by structural and/or functional studies have provided insights into the mechanisms by which the NER machinery recognizes and excises DNA lesions. Furthermore, we identify reported, but poorly characterized or unsubstantiated interactions in need of further validation. • NER is dynamic multistep pathway for the repair of bulky DNA lesions. • Protein-protein interaction regulate the steps in the NER reaction. • Structural and functional studies have elucidated the roles of protein-protein interactions in NER. • This review provides a guide to the protein-protein interactions in NER. [ABSTRACT FROM AUTHOR]

Dietary restriction (DR) is a universal anti-aging intervention, which reduces age-related nervous system pathologies and neurological decline. The degree to which the neuroprotective effect of DR operates by attenuating cell intrinsic degradative processes rather than influencing non-cell autonomous factors such as glial and vascular health or systemic inflammatory status is incompletely understood. Following up on our finding that DR has a remarkably large beneficial effect on nervous system pathology in whole-body DNA repair-deficient progeroid mice, we show here that DR also exerts strong neuroprotection in mouse models in which a single neuronal cell type, i.e., cerebellar Purkinje cells, experience genotoxic stress and consequent premature aging-like dysfunction. Purkinje cell specific hypomorphic and knock-out ERCC1 mice on DR retained 40 and 25% more neurons, respectively, with equal protection against P53 activation, and alike results from whole-body ERCC1-deficient mice. Our findings show that DR strongly reduces Purkinje cell death in our Purkinje cell-specific accelerated aging mouse model, indicating that DR protects Purkinje cells from intrinsic DNA-damage-driven neurodegeneration.

Beauveria bassiana serves as a main source of global fungal insecticides, which are based on the active ingredient of formulated conidia vulnerable to solar ultraviolet (UV) irradiation and restrained for all-weather application in green agriculture. The anti-UV proteins Rad1 and Rad10 are required for the nucleotide excision repair (NER) of UV-injured DNA in model yeast, but their anti-UV roles remain rarely exploredin filamentous fungi. Here, Rad1 and Rad10 orthologues that accumulated more in the nuclei than the cytoplasm of B. bassiana proved capable of reactivating UVB-impaired or UVB-inactivated conidia efficiently by 5h light exposure but incapable of doing so by 24 h dark incubation (NER) if the accumulated UVB irradiation was lethal. Each orthologue was found interacting with the other and two white collar proteins (WC1 and WC2), which proved to be regulators of two photolyases (Phr1 and Phr2) and individually more efficient in the photorepair of UVB-induced DNA lesions than either photolyase alone. The fungal photoreactivation activity was more or far more compromised when the protein–protein interactions were abolished in the absence of Rad1 or Rad10 than when either Phr1 or Phr2 lost function. The detected protein–protein interactions suggest direct links of either Rad1 or Rad10 to two photolyase regulators. In B. bassiana, therefore, Rad1 and Rad10 tied to the photolyase regulators have high activities in the photoprotection of formulated conidia from solar UV damage but insufficient NER activities in the field, where night (dark) time is too short, and no other roles in the fungal lifecycle in vitro and in vivo. [ABSTRACT FROM AUTHOR]

RPB1, the major and catalytic subunit of human RNA Polymerase II (RNAPII), is specifically degraded by the ubiquitin–proteasome system upon induction of DNA damage by different agents, such as ultraviolet (UV) light. The "last resort" model of RNAPII degradation states that a persistently stalled RNAPII is degraded at the site of the DNA lesion in order to facilitate access to Nucleotide Excision Repair (NER) factors, thereby promoting repair in template strands of active genes. Recent identification and mutation of the lysine residue involved in RPB1 ubiquitylation and degradation unveiled the relevance of RNAPII levels in the control of gene expression. Inhibition of RNAPII degradation after UV light exposure enhanced RNAPII loading onto chromatin, demonstrating that the mere concentration of RNAPII shapes the gene expression response. In this review, we discuss the role of RNAPII ubiquitylation in NER-dependent repair, recent advances in RPB1 degradation mechanisms and its consequences in gene expression under stress, both in normal and repair deficient cells. [ABSTRACT FROM AUTHOR]