Nucleotide Excision Repair Factors Research Articles

Persistent TFIIH binding to non-excised DNA damage causes cell and developmental failure

Congenital nucleotide excision repair (NER) deficiency gives rise to several cancer-prone and/or progeroid disorders. It is not understood how defects in the same DNA repair pathway cause different disease features and severity. Here, we show that the absence of functional ERCC1-XPF or XPG endonucleases leads to stable and prolonged binding of the transcription/DNA repair factor TFIIH to DNA damage, which correlates with disease severity and induces senescence features in human cells. In vivo, in C. elegans, this prolonged TFIIH binding to non-excised DNA damage causes developmental arrest and neuronal dysfunction, in a manner dependent on transcription-coupled NER. NER factors XPA and TTDA both promote stable TFIIH DNA binding and their depletion therefore suppresses these severe phenotypical consequences. These results identify stalled NER intermediates as pathogenic to cell functionality and organismal development, which can in part explain why mutations in XPF or XPG cause different disease features than mutations in XPA or TTDA.

Transcription-coupled repair of DNA–protein cross-links depends on CSA and CSB

Covalent DNA–protein cross-links (DPCs) are toxic DNA lesions that block replication and require repair by multiple pathways. Whether transcription blockage contributes to the toxicity of DPCs and how cells respond when RNA polymerases stall at DPCs is unknown. Here we find that DPC formation arrests transcription and induces ubiquitylation and degradation of RNA polymerase II. Using genetic screens and a method for the genome-wide mapping of DNA–protein adducts, DPC sequencing, we discover that Cockayne syndrome (CS) proteins CSB and CSA provide resistance to DPC-inducing agents by promoting DPC repair in actively transcribed genes. Consequently, CSB- or CSA-deficient cells fail to efficiently restart transcription after induction of DPCs. In contrast, nucleotide excision repair factors that act downstream of CSB and CSA at ultraviolet light-induced DNA lesions are dispensable. Our study describes a transcription-coupled DPC repair pathway and suggests that defects in this pathway may contribute to the unique neurological features of CS.

Transcription-coupled DNA–protein crosslink repair by CSB and CRL4CSA-mediated degradation

DNA–protein crosslinks (DPCs) arise from enzymatic intermediates, metabolism or chemicals like chemotherapeutics. DPCs are highly cytotoxic as they impede DNA-based processes such as replication, which is counteracted through proteolysis-mediated DPC removal by spartan (SPRTN) or the proteasome. However, whether DPCs affect transcription and how transcription-blocking DPCs are repaired remains largely unknown. Here we show that DPCs severely impede RNA polymerase II-mediated transcription and are preferentially repaired in active genes by transcription-coupled DPC (TC-DPC) repair. TC-DPC repair is initiated by recruiting the transcription-coupled nucleotide excision repair (TC-NER) factors CSB and CSA to DPC-stalled RNA polymerase II. CSA and CSB are indispensable for TC-DPC repair; however, the downstream TC-NER factors UVSSA and XPA are not, a result indicative of a non-canonical TC-NER mechanism. TC-DPC repair functions independently of SPRTN but is mediated by the ubiquitin ligase CRL4CSA and the proteasome. Thus, DPCs in genes are preferentially repaired in a transcription-coupled manner to facilitate unperturbed transcription.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia

BackgroundT-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. MethodsPediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. ResultsWe discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. ConclusionOur findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

The REV-ERB antagonist SR8278 modulates keratinocyte viability in response to UVA and UVB radiation.

The nucleotide excision repair (NER) system removes UV photoproducts from genomic DNA and is controlled by the circadian clock. Given that small-molecule compounds have been developed to target various clock proteins, we examined whether the cryptochrome inhibitor KS15 and REV-ERB antagonist SR8278 could modulate keratinocyte responses to UV radiation invitro. We observed that though SR8278 promoted cell viability in UVB-irradiated cells, it had little effect on NER or on the expression of the clock-regulated NER factor XPA. Rather, we found that both KS15 and SR8278 absorb light within the UV spectrum to limit initial UV photoproduct formation in DNA. Moreover, SR8278 promoted UVB viability even in cells in which the core circadian clock protein BMAL1 was disrupted, which indicates that SR8278 is likely acting via other REV-ERB transcriptional targets. We further observed that SR8278 sensitized keratinocytes to light sources containing primarily UVA wavelengths of light likely due to the generation of toxic reactive oxygen species. Though other studies have demonstrated beneficial effects of SR8278 in other model systems, our results here suggest that SR8278 has limited utility for UV photoprotection in the skin and will likely cause phototoxicity in humans or mammals exposed to solar radiation.

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes.

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage.

Treatment of Human HeLa Cells with Black Raspberry Extracts Enhances the Removal of DNA Lesions by the Nucleotide Excision Repair Mechanism.

As demonstrated by us earlier and by other researchers, a diet containing freeze-dried black raspberries (BRB) inhibits DNA damage and carcinogenesis in animal models. We tested the hypothesis that the inhibition of DNA damage by BRB is due, in part, to the enhancement of DNA repair capacity evaluated in the human HeLa cell extract system, an established in vitro system for the assessment of cellular DNA repair activity. The pre-treatment of intact HeLa cells with BRB extracts (BRBE) enhances the nucleotide excision repair (NER) of a bulky deoxyguanosine adduct derived from the polycyclic aromatic carcinogen benzo[a]pyrene (BP-dG) by ~24%. The NER activity of an oxidatively-derived non-bulky DNA lesion, guanidinohydantoin (Gh), is also enhanced by ~24%, while its base excision repair activity is enhanced by only ~6%. Western Blot experiments indicate that the expression of selected, NER factors is also increased by BRBE treatment by ~73% (XPA), ~55% (XPB), while its effects on XPD was modest (<14%). These results demonstrate that BRBE significantly enhances the NER yields of a bulky and a non-bulky DNA lesion, and that this effect is correlated with an enhancement of expression of the critically important NER factor XPA and the helicase XPB, but not the helicase XPD.

DNA Damage-Induced RNAPII Degradation and Its Consequences in Gene Expression.

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.

TRF1 uses a noncanonical function of TFIIH to promote telomere replication.

Telomeric DNA challenges the replisome and requires TRF1 for efficient duplication. TRF1 recruits the BLM helicase, but BLM loss does not explain the extensive telomere fragility, ATR signaling, and sister telomere associations (STAs) induced by TRF1 deletion. Here, we document that Helix2 of the TRFH domain and Helix1 of the Myb domain of TRF1 are required for efficient telomere replication. Mutation of both helices generated a TRF1 separation-of-function mutant (TRF1-E83K/LW-TI) that induced severe telomere replication defects but no ATR signaling or STAs. We identified the transcription and nucleotide excision repair (NER) factor TFIIH as a critical effector of TRF1. Loss of TFIIH subunits, but no other NER factors, caused the same telomere replication phenotypes as the TRF1-E83K/LW-TI mutant independent of the effects on TRF1 expression. TFIIH subunits coimmunoprecipitated with wild-type TRF1 but not with TRF1-E83K/LW-TI. These results establish that the major mechanism by which TRF1 ensures telomere replication involves a noncanonical function of TFIIH.

604 RNA methylation facilitates the repair of UV-induced DNA damage and suppresses photocarcinogenesis

UV-induced DNA damage is repaired by nucleotide excision repair (NER) that corrects bulky helix-distorting DNA lesions across the whole genome and is essential for preventing mutagenesis and skin cancer. Here we show that METTL14 (methyltransferase-like 14), a critical component of the m6A RNA methyltransferase complex, promotes the repair of UV-induced DNA damage through regulating m6A mRNA methylation-mediated translation of the NER factor DDB2 and suppresses UV-induced skin tumorigenesis. Ultraviolet irradiation down-regulates METTL14 protein through NBR1-dependent selective autophagy.

Identifying Biomarkers of Cisplatin Sensitivity in Non-Small Cell Lung Cancer via Comprehensive Integrative Analysis

Background: Only 30-40% of non-small cell lung cancer (NSCLC) patients are clinically sensitive to cisplatin-based chemotherapy. Thus, it is necessary to identify biomarkers for personalized cisplatin chemotherapy in NSCLC. However, data heterogeneity and low-value density make it challenging to detect reliable cisplatin efficacy biomarkers using traditional analysis methods. Objective: This paper aims to find reliable cisplatin efficacy biomarkers for NSCLC patients using comprehensive integrative analysis. Method: We searched online resources and collected six NSCLC transcriptomics data sets with responses to cisplatin. The six data sets are divided into two groups: the learning group for biomarker identification and the test group for independent validation. We performed comprehensive integrative analysis under two kinds of frameworks, i.e., one-level and two-level, with three integrative models. Pathway analysis was performed to estimate the biological significance of the resulting biomarkers. For independent validation, logrank statistic was employed to test how significant the difference of Kaplan- Meier (KM) curves between two patient groups is, and the Cox proportional-hazards model was used to test how the expression of a gene is associated with patients’ survival time. Especially, a permutation test was performed to verify the predictive power of a biomarker panel on cisplatin efficacy. For comparison, we also analyzed each learning data set individually, in which three popular differential expression models, Limma, SAM, and RankSum, were used. Results: A total of 318 genes were identified as a core panel of cisplatin efficacy markers for NSCLC patients, exhibiting consistent differential expression between cisplatin-sensitive and –resistant groups across studies. A total of 129 of 344 KEGG pathways were found to be enriched in the core panel, reflecting a picture of the molecular mechanism of cisplatin resistance in NSCLC. By mapping onto the KEGG pathway tree, we found that a KEGG pathway-level I module, genetic information processing, is most active in the core panel with the highest activity ratio in response to cisplatin in NSCLC as expected. Related pathways include mismatch repair, nucleotide excision repair, aminoacyl-tRNA biosynthesis, and basal transcription factors, most of which respond to DNA double-strand damage in patients. Evaluation on two independent data sets demonstrated the predictive power of the core marker panel for cisplatin sensitivity in NSCLC. Also, some single markers, e.g., MST1R, were observed to be remarkably predictive of cisplatin resistance in NSCLC. Conclusion: Integrative analysis is more powerful in detecting biomarkers for cisplatin efficacy by overcoming data heterogeneity and low-value density in data sets, and the identified core panel (318 genes) can help develop personalized medicine of cisplatin chemotherapy for NSCLC patients.

Nucleotide excision repair: a versatile and smart toolkit.

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.

Flavonoid Nobiletin Exhibits Differential Effects on Cell Viability in Keratinocytes Exposed to UVA versus UVB Radiation.

The polymethoxylated flavonoid nobiletin has been shown to suppress inflammatory responses to UVB radiation and to enhance circadian rhythms. Because expression of the core nucleotide excision repair (NER) factor XPA and the rate of removal of UV photoproducts from DNA are regulated by the circadian clock, we investigated whether the beneficial effects of nobiletin in UVB-exposed cells could be due in part to enhanced NER. Although nobiletin limited UVB-irradiated human keratinocytes from undergoing cell death, we found that this enhanced survival was not associated with increased NER or XPA expression. Instead, nobiletin reduced initial UV photoproduct formation and promoted a G1 cell cycle arrest. We then examined the implications of this findings for exposures to solar radiation through use of a solar simulated light (SSL) source that contains primarily UVA radiation. In striking contrast to the results obtained with UVB radiation, nobiletin instead sensitized keratinocytes to both the SSL and a more defined UVA radiation source. This enhanced cell death was correlated with a photochemical change in nobiletin absorption spectrum and the production of reactive oxygen species. We conclude that nobiletin is unlikely to be a useful compound for protecting keratinocytes against the harmful effects of solar UV radiation.

A chromosome-level genome of the human blood fluke Schistosoma japonicum identifies the genomic basis of host-switching.

The evolution and adaptation of S.japonicum, a zoonotic parasite that causes human schistosomiasis, remain unclear because of the lack of whole-genome data. We construct a chromosome-level S.japonicum genome and analyze it together with 72 samples representing six populations of the entire endemic region. We observe a Taiwan zoophilic lineage splitting from zoonotic populations ∼45,000 years ago, consistent with the divergent history of their intermediate hosts. Interestingly, we detect a severe population bottleneck in S.japonicum, largely coinciding with human history in Asia during the last glacial maximum. We identify several genomic regions underlying natural selection, including GATAD2A and Lmln, both showing remarkable differentiation among different areas. RNAi knockdown suggests association of GATAD2A with parasite development and infection in definitive hosts, while Lmln relates to the specificity of the intermediate hosts. Our study provides insights into the evolution of S.japonicum and serves as a resource for further studies.

Camptothecin compromises transcription recovery and cell survival against cisplatin and ultraviolet irradiation regardless of transcription-coupled nucleotide excision repair

DNA-damaging anti-cancer drugs are used clinically to induce cell death by causing DNA strand breaks or DNA replication stress. Camptothecin (CPT) and cisplatin are commonly used anti-cancer drugs, and their combined use enhances the anti-tumour effects. However, the mechanism underlying this enhanced effect has not been well studied. In this study, we analysed the combined effect of CPT and cisplatin or ultraviolet (UV) and found that CPT suppresses transcription recovery after UV damage and induces the disappearance of the Cockayne syndrome group B (CSB) protein, a transcription-coupled nucleotide excision repair (TC-NER) factor. This CPT-induced disappearance of CSB expression was suppressed by proteasome and transcription inhibitors. Moreover, CSB ubiquitination was detected after CPT treatment in a transcription-dependent manner, suggesting that the transcription stress caused by CPT induces CSB ubiquitination, resulting in CSB undetectability. However, Cockayne syndrome group A (CSA) and CUL4A were not involved in the CPT-induced CSB undetectability, suggesting that CSB ubiquitination caused by CPT is regulated differently from the UV response. However, cisplatin or UV sensitivity was enhanced by CPT even in CSB- or CSA-knockout cells. Furthermore, the excessive CSB expression, which suppressed CSB ubiquitination, did not cancel the combined effect of CPT. These results suggest that CPT blocks the repair of cisplatin or UV-induced DNA damage regardless of TC-NER status. CPT possibly compromised the alternative repair pathways other than TC-NER, leading to the suppression of transcription recovery and enhancement of cell killing.

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

Nucleotide excision repair (NER) counteracts the onset of cancer and aging by removing helix-distorting DNA lesions via a "cut-and-patch"-type reaction. The regulatory mechanisms that drive NER through its successive damage recognition, verification, incision, and gap restoration reaction steps remain elusive. Here, we show that the RAD5-related translocase HLTF facilitates repair through active eviction of incised damaged DNA together with associated repair proteins. Our data show a dual-incision-dependent recruitment of HLTF to the NER incision complex, which is mediated by HLTF's HIRAN domain that binds 3'-OH single-stranded DNA ends. HLTF's translocase motor subsequently promotes the dissociation of the stably damage-bound incision complex together with the incised oligonucleotide, allowing for an efficient PCNA loading and initiation of repair synthesis. Our findings uncover HLTF as an important NER factor that actively evicts DNA damage, thereby providing additional quality control by coordinating the transition between the excision and DNA synthesis steps to safeguard genome integrity.

Pharmacological inhibition of cryptochrome and REV-ERB promotes DNA repair and cell cycle arrest in cisplatin-treated human cells

Nucleotide excision repair (NER) and cell cycle checkpoints impact the ability of the anti-cancer drug cisplatin to inhibit cell proliferation and induce cell death. Genetic studies have shown that both NER and cell cycle progression are impacted by the circadian clock, which has emerged as a novel pharmacological target for the treatment of various disease states. In this study, cultured human cell lines were treated with combinations of cisplatin and the circadian clock modulating compounds KS15 and SR8278, which enhance circadian clock transcriptional output by inhibiting the activities of the cryptochrome and REV-ERB proteins, respectively. Treatment of cells with KS15 and SR8278 protected cells against the anti-proliferative effects of cisplatin and increased the expression of NER factor XPA and cell cycle regulators Wee1 and p21 at the mRNA and protein level. Correlated with these molecular changes, KS15 and SR8278 treatment resulted in fewer unrepaired cisplatin–DNA adducts in genomic DNA and a higher fraction of cells in the G1 phase of the cell cycle. Thus, the use of pharmacological agents targeting the circadian clock could be a novel approach to modulate the responses of normal and cancer cells to cisplatin chemotherapy regimens.

Analysis of the PcrA-RNA polymerase complex reveals a helicase interaction motif and a role for PcrA/UvrD helicase in the suppression of R-loops.

The PcrA/UvrD helicase binds directly to RNA polymerase (RNAP) but the structural basis for this interaction and its functional significance have remained unclear. In this work, we used biochemical assays and hydrogen-deuterium exchange coupled to mass spectrometry to study the PcrA-RNAP complex. We find that PcrA binds tightly to a transcription elongation complex in a manner dependent on protein:protein interaction with the conserved PcrA C-terminal Tudor domain. The helicase binds predominantly to two positions on the surface of RNAP. The PcrA C-terminal domain engages a conserved region in a lineage-specific insert within the β subunit which we identify as a helicase interaction motif present in many other PcrA partner proteins, including the nucleotide excision repair factor UvrB. The catalytic core of the helicase binds near the RNA and DNA exit channels and blocking PcrA activity in vivo leads to the accumulation of R-loops. We propose a role for PcrA as an R-loop suppression factor that helps to minimize conflicts between transcription and other processes on DNA including replication.

Strand-specific effect of Rad26 and TFIIS in rescuing transcriptional arrest by CAG trinucleotide repeat slip-outs

Transcription induced CAG repeat instability is associated with fatal neurological disorders. Genetic approaches found transcription-coupled nucleotide excision repair (TC-NER) factor CSB protein and TFIIS play critical roles in modulating the repeat stability. Here, we took advantage of an in vitro reconstituted yeast transcription system to investigate the underlying mechanism of RNA polymerase II (Pol II) transcriptional pausing/stalling by CAG slip-out structures and the functions of TFIIS and Rad26, the yeast ortholog of CSB, in modulating transcriptional arrest. We identified length-dependent and strand-specific mechanisms that account for CAG slip-out induced transcriptional arrest. We found substantial R-loop formation for the distal transcriptional pausing induced by template strand (TS) slip-out, but not non-template strand (NTS) slip-out. In contrast, Pol II backtracking was observed at the proximal transcriptional pausing sites induced by both NTS and TS slip-out blockage. Strikingly, we revealed that Rad26 and TFIIS can stimulate bypass of NTS CAG slip-out, but not TS slip-out induced distal pausing. Our biochemical results provide new insights into understanding the mechanism of CAG slip-out induced transcriptional pausing and functions of transcription factors in modulating transcription-coupled CAG repeat instability, which may pave the way for developing potential strategies for the treatment of repeat sequence associated human diseases.

M6A RNA Methylation Regulators Act as Potential Prognostic Biomarkers in Lung Adenocarcinoma

N6-methyladenosine [m(6)A/m6A] methylation is one of the most common RNA modifications in eukaryotic cell mRNA and plays an important regulatory role in mRNA metabolism, splicing, translocation, stability, and translation. Previous studies have demonstrated that the m6A modification is highly associated with tumor cell proliferation, migration, and invasion. In the present study, five m6A regulatory factors have been revealed, namely heterogeneous nuclear ribonucleoprotein A2/B1(HNRNPA2B1), heterogeneous nuclear ribonucleoprotein C (HNRNPC), Vir like m6A methyltransferase associated protein (KIAA1429/VIRMA), RNA binding motif protein 15 (RBM15) and methyltransferase like 3 (METTL3), which are closely related to the overall survival (OS) of patients with lung adenocarcinoma (LUAD). These five m6A regulatory factors exhibited potential prognostic value for the 1, 3, and 5-years survival outcomes of LUAD patients. Our findings revealed that several signaling pathways, such as cell cycle, DNA replication, RNA degradation, RNA polymerase, nucleotide excision repair and basal transcription factors, are activated in the high-risk group of LUAD patients.