Search Results (28)

DNA interstrand cross-links (ICLs) are among the most cytotoxic forms of DNA damage, arising when the two strands of the DNA helix are covalently linked by crosslink-inducing agents such as bifunctional alkylating agents and reactive aldehydes. Several studies have demonstrated that ICLs can also be induced by reactive oxygen and nitrogen species. We previously reported that under oxidative conditions, the major oxidative adenine lesion 7,8-dihydro-8-oxoadenine (oxoA) can efficiently generate a novel class of oxoA-G ICLs, structurally resembling guanine–guanine (G–G) cross-links that can be induced by reactive nitrogen species. To investigate the mutagenic potential of these oxidation-induced ICLs in cells, we employed a SupF-based mutagenesis assay using bacterial cells. A single site-specific oxoA–G ICL was synthesized and incorporated into a plasmid, which was then introduced into an E. coli reporter strain to assess mutation profiles induced by both oxoA and oxoA–G ICLs. Our results show that oxoA–G ICLs cause A-to-C/T and G-to-C transversion mutations at the oxoA-G cross-link site, demonstrating highly promutagenic nature of the lesion in bacterial cells. We propose that the oxoA–G ICL may promote transversion mutations, likely driven by a syn conformer of unhooked oxoA-G ICL repair intermediates during translesion synthesis. Full article

Background/Objectives: DNA interstrand cross-links (ICLs) mark one of the most deleterious lesions that can preclude strand separation required for essential cellular processes. Efforts to discover ICL-inducing agents and endogenous substrates for ICL repair pathways have led to the identification of structurally diverse ICLs produced by reactive aldehydes and abasic sites, among others. While several studies point to UV rays as ICL-inducing agents, UV ray-induced ICL formation from biologically relevant DNA lesions has been rarely reported. We conjectured that solar radiation-induced reactive oxygen species may give rise to ICLs via further oxidation of DNA lesions with lower redox potential (e.g., 8-oxoadenine (oxoA)). Here, we present the discovery of ICL production via light-induced modification of the major oxidative adenine lesion oxoA. Methods/Results: In the absence of a photosensitizer, both UVC and UVB rays, but not UVA and visible rays, trigger the formation of oxoA-G ICLs, albeit in low yields. By contrast, the inclusion of the naturally occurring photosensitizer riboflavin in the cross-linking reaction makes UVA and visible rays readily generate oxoA-G ICLs, suggesting solar radiation facilitates the formation of oxoA ICLs in vivo. Conclusions: The plausible oxoA-G ICL formation mechanism concerns the further oxidation of oxoA into an iminoquinone, followed by the nucleophilic attack of the opposite guanine on the iminoquinone. OxoA-G ICLs represent rare examples of ICLs produced by photosensitization. These results will contribute to the discovery of a novel form of ICLs induced by solar radiation. Full article

The DNA Damage Response (DDR) network is an essential machinery for maintaining genomic integrity, with DDR defects being implicated in cancer initiation, progression, and treatment resistance. Moreover, oxidative stress, an imbalance between reactive oxygen species production and antioxidant defense, can significantly impact cell viability, leading to cell death or survival. Herein, we tested the hypothesis that DDR-related signals and redox status measured in multiple myeloma (MM) cell lines correlate with the sensitivity to genotoxic insults. At baseline and following irradiation with Ultraviolet C (UVC; 50 J/m²) or treatment with melphalan (100 μg/mL for 5 min) DDR-related parameters, redox status expressed as GSH/GSSG ratio and apurinic/apyrimidinic sites were evaluated in a panel of eleven human MM cell lines and one healthy B lymphoblastoid cell line. We found that MM cell lines with increased apoptosis rates displayed significantly higher levels of endogenous/baseline DNA damage, reduced GSH/GSSG ratio, augmented apurinic/apyrimidinic lesions, decreased nucleotide excision repair and interstrand crosslinks repair capacities, and highly condensed chromatin structure. Taken together, these findings demonstrate that DDR-related parameters and redox status correlate with the sensitivity of MM cells to DNA-damaging agents, specifically melphalan, and, if further validated, may be exploited as novel sensitive/effective biomarkers. Full article

Fanconi anemia (FA) is a genetic disorder characterized by congenital anomalies, bone marrow failure, and cancer predisposition. Among other solid cancers, head and neck squamous cell carcinoma (FA HNSCC) is the most common cancer type in individuals with FA. The FA pathway is required for the complete repair of DNA interstrand crosslinks (ICLs), and unresolved ICLs result in cell cycle arrest, apoptosis, or complex chromosomal rearrangements due to chromosome breaks, ultimately leading to tumorigenesis. FA HNSCCs present earlier (median age of onset in the 30s) and exhibit a more aggressive course with frequent recurrence and second primaries, and entail a poorer survival rate compared to sporadic HNSCC. FA HNSCCs are mostly human papillomavirus (HPV)-negative and frequently carry somatic copy number variations (CNVs), which amplify oncogenes implicated in sporadic HNSCC, but single-nucleotide variants or small insertions and deletions are less frequent than in HPV-negative sporadic HNSCC. A subset of sporadic HNSCC carries pathogenic mutations or promoter methylation in FA genes, which also harbor characteristic somatic CNVs, suggesting shared molecular underpinnings with FA HNSCC. Heightened inflammation from genomic instability and transcriptional activation of retrotransposons contribute to tumorigenesis and increased invasiveness by the epithelial-to-mesenchymal transition. Due to heightened sensitivity to DNA crosslinking agents in patients with FA, platinum-based chemotherapy is generally avoided, which presents a significant hurdle for treatment and thereby leaves limited therapeutic options. Surgical management is the mainstay of therapy if possible, and targeted therapy has been increasingly studied in HNSCC in FA. Full article

Background/Objectives: DNA-damaging agents can contribute to genetic instability, and such agents are often used in cancer chemotherapeutic regimens due to their cytotoxicity. Thus, understanding the mechanisms involved in DNA damage processing can not only enhance our knowledge of basic DNA repair mechanisms but may also be used to develop improved chemotherapeutic strategies to treat cancer. The high-mobility group box protein 1 (HMGB1) is a known nucleotide excision repair (NER) cofactor, and its family member HMGB3 has been implicated in chemoresistance in ovarian cancer. Here, we aim to understand the potential role(s) of HMGB3 in processing DNA damage. Methods: A potential role in NER was investigated using HMGB3 knockout human cell lines in response to UV damage. Subsequently, potential roles in DNA interstrand crosslink (ICL) and DNA double-strand break (DSB) repair were investigated using mutagenesis assays, metaphase spreads, foci formation, a variety of DNA repair assays, and TagSeq analyses in human cells. Results: Interestingly, unlike HMGB1, HMGB3 does not appear to play a role in NER. We found evidence to suggest that HMGB3 is involved in the processing of both DSBs and ICLs in human cells. Conclusions: These novel results elucidate a role for HMGB3 in DNA damage repair and, surprisingly, also indicate a distinct role of HMGB3 in DNA damage repair from that of HMGB1. These findings advance our understanding of the role of HMGB3 in chemotherapeutic drug resistance and as a target for new chemotherapeutic strategies in the treatment of cancer. Full article

Background/Objectives: DNA damage response (DDR) is a network of molecular pathways associated with the pathogenesis and progression of several diseases, as well as the outcome of chemotherapy. Moreover, the intracellular redox status is essential for maintaining cell viability and controlling cellular signaling. Herein, we analyzed DDR signals and redox status in peripheral blood mononuclear cells (PBMCs) from patients with lung cancer with different response rates to platinum-based chemotherapy. Methods: Several DDR-associated signals and redox status, expressed as the GSH/GSSG ratio, were measured in two lung cancer cell lines (A549, H1299), two normal fibroblast cell lines (WS1, 1BR3hT), and PBMCs from 20 healthy controls and 32 patients with lung cancer at baseline (17 responders and 15 non-responders to subsequent platinum-based chemotherapy). Results: Higher levels of endogenous/baseline DNA damage, decreased GSH/GSSG ratios, and augmented apurinic/apyrimidinic sites, as well as lower nucleotide excision repair (NER) and increased interstrand cross-links (ICLs) repair efficiencies, were observed in lung cancer cell lines compared with normal ones (all p < 0.05). Moreover, PBMCs from patients with lung cancer showed reduced GSH/GSSG ratios, augmented apurinic/apyrimidinic sites, decreased NER and ICL repair capacities, and lower apoptosis rates, compared with healthy controls (all p < 0.001). Interestingly, PBMCs from patients who are responders are characterized by reduced GSH/GSSG ratios, augmented apurinic/apyrimidinic sites, decreased NER and ICL repair capacities, and higher apoptosis rates compared with patients who are non-responders (all p < 0.01). Conclusions: Together, DDR-associated parameters and redox status measured in PBMCs from patients with lung cancer at baseline are associated with the therapeutic benefit of platinum-based chemotherapy. Full article

DNA replication represents a series of precisely regulated events performed by a complex protein machinery that guarantees accurate duplication of the genetic information. Since DNA replication is permanently faced by a variety of exogenous and endogenous stressors, DNA damage response, repair and replication must be closely coordinated to maintain genomic integrity. HROB has been identified recently as a binding partner and activator of the Mcm8/9 helicase involved in DNA interstrand crosslink (ICL) repair. We identified HROB independently as a nuclear protein whose expression is co-regulated with various DNA replication factors. Accordingly, the HROB protein level showed a maximum in S phase and a downregulation in quiescence. Structural prediction and homology searches revealed that HROB is a largely intrinsically disordered protein bearing a helix-rich region and a canonical oligonucleotide/oligosaccharide-binding-fold motif that originated early in eukaryotic evolution. Employing a flow cytometry Förster resonance energy transfer (FRET) assay, we detected associations between HROB and proteins of the DNA replication machinery. Moreover, ectopic expression of HROB protein led to an almost complete shutdown of DNA replication. The available data imply a function for HROB during DNA replication across barriers such as ICLs. Full article

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair. Full article

The accumulation of oxidative DNA base damage can severely disrupt the integrity of the genome and is strongly associated with the development of cancer. DNA glycosylase is the critical enzyme that initiates the base excision repair (BER) pathway, recognizing and excising damaged bases. The Nei endonuclease VIII-like 3 (NEIL3) is an emerging DNA glycosylase essential in maintaining genome stability. With an in-depth study of the structure and function of NEIL3, we found that it has properties related to the process of base damage repair. For example, it not only prefers the base damage of single-stranded DNA (ssDNA), G-quadruplex and DNA interstrand crosslinks (ICLs), but also participates in the maintenance of replication fork stability and telomere integrity. In addition, NEIL3 is strongly associated with the progression of cancers and cardiovascular and neurological diseases, is incredibly significantly overexpressed in cancers, and may become an independent prognostic marker for cancer patients. Interestingly, circNEIL3, a circular RNA of exon-encoded origin by NEIL3, also promotes the development of multiple cancers. In this review, we have summarized the structure and the characteristics of NEIL3 to repair base damage. We have focused on NEIL3 and circNEIL3 in cancer development, progression and prognosis. Full article

Genetic instability can result from increases in DNA damage and/or alterations in DNA repair proteins and can contribute to disease development. Both exogenous and endogenous sources of DNA damage and/or alterations in DNA structure (e.g., non-B DNA) can impact genome stability. Multiple repair mechanisms exist to counteract DNA damage. One key DNA repair protein complex is ERCC1-XPF, a structure-specific endonuclease that participates in a variety of DNA repair processes. ERCC1-XPF is involved in nucleotide excision repair (NER), repair of DNA interstrand crosslinks (ICLs), and DNA double-strand break (DSB) repair via homologous recombination. In addition, ERCC1-XPF contributes to the processing of various alternative (i.e., non-B) DNA structures. This review will focus on the processing of alternative DNA structures by ERCC1-XPF. Full article

Outdoor air pollution is a mixture of multiple atmospheric pollutants, among which nitrogen oxide (NOx) stands out due to its association with several diseases. NOx reactivity can conduct to DNA damage as severe as interstrand crosslinks (ICL) formation, that in turn is able to block DNA replication and transcription. Experimental studies have suggested that the ICL formation due to NOx is realized through a diazonium intermediate (DI). In this work, we have modeled the DI structure, including a DNA double-strand composed of two base pairs GC/CG, being diazotized as one of the guanine nucleotides. The structural stability of DNA with DI lesion was essayed through 500 ns molecular dynamics simulations. It was found that the DNA structure of the oligonucleotide is stable when the DI is present since the loss of a Guanine–Cytosine hydrogen bond is replaced by the presence of two cation-π interactions. Additionally, we have studied the mechanism of formation of a crosslink between the two guanine nucleobases from the modeled DI by carrying out DFT calculations at the M06-L/DNP+ level of theory. Our results show that the mechanism is thermodynamically favored by a strong stabilization of the ICL product, and the process is kinetically viable since its limiting stage is accessible. Full article

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy. Full article

Fanconi anaemia (FA)-related proteins function in interstrand crosslink (ICL) repair pathways and multiple damage repair pathways. Recent studies have found that FA proteins are involved in the regulation of replication stress (RS) in alternative lengthening of telomeres (ALT). Since ALT cells often exhibit high-frequency ATRX mutations and high levels of telomeric secondary structure, high levels of DNA damage and replicative stress exist in ALT cells. Persistent replication stress is required to maintain the activity of ALT mechanistically, while excessive replication stress causes ALT cell death. FA proteins such as FANCD2 and FANCM are involved in the regulation of this balance by resolving or inhibiting the formation of telomere secondary structures to stabilize stalled replication forks and promote break-induced repair (BIR) to maintain the survival of ALT tumour cells. Therefore, we review the role of FA proteins in replication stress in ALT cells, providing a rationale and direction for the targeted treatment of ALT tumours. Full article

The Fanconi anemia (FA) DNA repair pathway coordinates a faithful repair mechanism for stalled DNA replication forks caused by factors such as DNA interstrand crosslinks (ICLs) or replication stress. An important role of FA pathway activation is initiated by monoubiquitination of FANCD2 and its binding partner of FANCI, which is regulated by the ATM-related kinase, ATR. Therefore, regulation of the FA pathway is a good example of the contribution of ATR to genome stability. In this short review, we summarize the knowledge accumulated over the years regarding how the FA pathway is activated via phosphorylation and monoubiquitination. Full article

Furan is a volatile compound that is formed in foods during thermal processing. It is classified as a possible human carcinogen by international authorities based on sufficient evidence of carcinogenicity from studies in experimental animals. Although a vast number of studies both in vitro and in vivo have been performed to investigate furan genotoxicity, the results are inconsistent, and its carcinogenic mode of action remains to be clarified. Here, we address the mutagenic and clastogenic activity of furan and its prime reactive metabolite cis-2 butene-1,4-dial (BDA) in mammalian cells in culture and in mouse animal models in a search for DNA lesions responsible of these effects. To this aim, Fanconi anemia-derived human cell lines defective in the repair of DNA inter-strand crosslinks (ICLs) and Ogg1^−/− mice defective in the removal of 8-hydroxyguanine from DNA, were used. We show that both furan and BDA present a weak (if any) mutagenic activity but are clear inducers of clastogenic damage. ICLs are strongly indicated as key lesions for chromosomal damage whereas oxidized base lesions are unlikely to play a critical role. Full article