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LYAR (Ly-1 antibody reactive) is a transcription factor with a specific DNA-binding domain, which plays a key role in the regulation of embryonic stem cell self-renewal and differentiation. However, the role of LYAR in human cancers remains unclear. This study aimed to analyze the prognostic value of LYAR in cancer. In this study, we evaluated the prognostic value of LYAR in various tumors. We research found that, compared with normal tissues, LYAR levels werehigher in a variety of tumors. LYAR expression level was associated with poor overall survival, progression-free interval, and disease-specific survival. LYAR expression was also related to tumor grade, stage, age, and tumor status. Cell counting kit-8, Transwell, and wound healing assay showed that knocking out LYAR significantly inhibited the proliferation, migration, and invasion of hepatocellular carcinoma cells. In addition, this study found that LYARexpression was significantly positively correlated with MKI67IP, BZW2, and CCT2. Gene set enrichment analysis results showed that samples with high LYAR expression levels were rich in spliceosomes, RNA degradation, pyrimidine metabolism, cell cycle, nucleotide excision repair, and base excision repair.

Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and ß-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTLH1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a lower binding affinity for lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3–6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a−/− mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a−/− mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis. FAM72A differentially controls mutation rates by regulating uracil processing at different stages of the cell cycle, thereby regulating somatic hypermutation and class-switch recombination in B cells.

Poly(ADP-ribosyl)ation catalyzed by poly(ADP-ribose) polymerases (PARPs) is one of the immediate cellular responses to DNA damage. The histone PARylation factor 1 (HPF1) discovered recently to form a joint active site with PARP1 and PARP2 was shown to limit the PARylation activity of PARPs and stimulate their NAD+-hydrolase activity. Here we demonstrate that HPF1 can stimulate the DNA-dependent and DNA-independent autoPARylation of PARP1 and PARP2 as well as the heteroPARylation of histones in the complex with nucleosome. The stimulatory action is detected in a defined range of HPF1 and NAD+ concentrations at which no HPF1-dependent enhancement in the hydrolytic NAD+ consumption occurs. PARP2, comparing with PARP1, is more efficiently stimulated by HPF1 in the autoPARylation reaction and is more active in the heteroPARylation of histones than in the automodification, suggesting a specific role of PARP2 in the ADP-ribosylation-dependent modulation of chromatin structure. Possible role of the dual function of HPF1 in the maintaining PARP activity is discussed., Kurgina et al. conduct in vitro characterization of the effect of HPF1 on the catalytic output of PARP1 and PARP2 under several experimental conditions. The authors report that the DNAdependent and DNA-independent autoPARylation of PARP1 and PARP2, as well as the heteroPARylation of histones in complex with the nucleosome are stimulated by HPF1 in a certain range of HPF1 and NAD + concentrations.

There is an increase in the incidence of early onset colorectal carcinoma (EOCRC). To better understand if there is any difference in molecular pathogenesis of EOCRC and late onset colorectal carcinoma (LOCRC), we compared the clinical, histological, transcriptome, and methylome profile of paired CRC and healthy colonic tissue from 67 EOCRC and 98 LOCRC patients. The frequency of stage 3 CRC, lymph node involvement, lymphovascular invasion, and perineural invasion was higher in the EOCRC group. Many of the cancer related pathways were differentially expressed in CRC tissue in both EOCRC and LOCRC patients. However, the magnitude of differential expression for some groups of genes, such as DNA damage repair genes and replication stress genes, were significantly less pronounced in the EOCRC group, suggesting less efficient DNA damage repair to be associated with EOCRC. A more marked methylation of “growth factor receptor” genes in LOCRC correlated with a more pronounced down-regulation of those genes in that group. From a therapeutic point of view, more over-expression of fatty acid synthase (FASN) among the LOCRC patients may suggest a better response of FASN targeted therapy in that group. The age of onset of CRC did not appear to modify the response of cis-platin or certain immune checkpoint inhibitors. We found some differences in the molecular pathogenesis in EOCRC and LOCRC that may have some biological and therapeutic significance.

Online ahead of print.; International audience; Background: The generation of DNA damage by ultra-violet radiations (UV) is well established, and both the nature of the DNA lesions and their respective DNA repair pathways have largely been described. Besides UV rays, visible light constitutes a very important part of the sun spectrum where blue light is considered a significant contributor to premature aging. However, blue light-induced DNA damage has not been deeply explored yet.Methods: In the present study, we assessed in human skin keratinocytes the DNA and chromosome damaging activities of blue light rays (415 nm) as well as their associated DNA repair mechanisms.Results: Our results demonstrated that blue light induced dose-dependent DNA damage in human keratinocytes. Both oxidative and cyclobutane-pyrimidine-dimer (CPD) DNA lesions were generated. They were repaired through base excision repair (BER) and nucleotide excision repair (NER) pathways, respectively. Moreover, by using the micronucleus assay we demonstrated, for the first time, that a blue wavelength exerted a clastogenic/aneugenic effect in human keratinocytes, leading to chromosome aberration.Conclusion: We concluded that, in normal human keratinocytes, blue light creates genotoxic lesions which might accelerate or at least contribute to premature skin aging.Keywords: DNA damage; DNA repair; blue light; keratinocytes.© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Genotoxicity testing plays an important role in the safety assessment of pharmaceuticals, pesticides and chemical substances. Among the guidelines for various genotoxicity tests, the in vitro genotoxicity test battery comprises the bacterial Ames test and mammalian cell assays. Several chemicals exhibit conflicting results for the bacterial Ames test and mammalian cell genotoxicity studies, which may stem from the differences in DNA repair capacity or metabolism, between different cell types or species. For better understanding the mechanistic implications regarding conflict outcomes between different assay systems, it is necessary to develop in vitro genotoxicity testing approaches with higher specificity towards DNA-damaging reagents. We have recently established an improved thymidine kinase (TK) gene mutation assay (TK assay) i.e. deficient in DNA excision repair system using human lymphoblastoid TK6 cells lacking XRCC1 and XPA (XRCC1−/−/XPA−/−), the core factors of base excision repair (BER) and nucleotide excision repair (NER), respectively. This DNA repair-deficient TK6 cell line is expected to specifically evaluate the genotoxic potential of chemical substances based on the DNA damage. We focussed on four reagents, N-(1-naphthyl)ethylenediamine dihydrochloride (NEDA), p-phenylenediamine (PPD), auramine and malachite green (MG) as the Ames test-positive chemicals. In our assay, assessment using XRCC1−/−/XPA−/− cells revealed no statistically significant increase in the mutant frequencies after treatment with NEDA, PPD and MG, suggesting the chemicals to be non-genotoxic in humans. The observations were consistent with that of the follow-up in vivo studies. In contrast, the mutant frequency was markedly increased in XRCC1−/−/XPA−/− cells after treatment with auramine. The results suggest that auramine is the genotoxic reagent that preferentially induces DNA damages resolved by BER and/or NER in mammals. Taken together, BER/NER-deficient cell-based genotoxicity testing will contribute to elucidate the mechanism of genotoxicity and therefore play a pivotal role in the accurate safety assessment of chemical substances.

Summary Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes “trapped” on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1−/− cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an “anti-trapper” that prevents toxic PARP1 activity., Graphical abstract, Highlights • XRCC1 prevents endogenous PARP1 trapping during DNA base excision repair • PARP1 trapping impedes base excision repair and increases sensitivity to base damage • In the absence of PARP1, XRCC1 is dispensable for DNA base excision repair, Demin et al. show that the essential role of the scaffold protein XRCC1 during DNA base excision repair is to prevent toxic “trapping” of PARP1 on SSB intermediates, which otherwise block this essential repair process and lead to increased cellular sensitivity to DNA base damage.

G:T mismatches, the major mispairs generated during DNA metabolism, are repaired in part by mismatch-specific DNA glycosylases such as methyl-CpG-binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). Mismatch-specific DNA glycosylases must discriminate the mismatches against million-fold excess correct base pairs. MBD4 efficiently removes thymine opposite guanine but not opposite adenine. Previous studies have revealed that the substrate thymine is flipped out and enters the catalytic site of the enzyme, while the estranged guanine is stabilized by Arg468 of MBD4. To gain further insights into the mismatch discrimination mechanism of MBD4, we assessed the glycosylase activity of MBD4 toward various base pairs. In addition, we determined a crystal structure of MBD4 bound to T:O6-methylguanine-containing DNA, which suggests the O6 and N2 of purine and the O4 of pyrimidine are required to be a substrate for MBD4. To understand the role of the Arg468 finger in catalysis, we evaluated the glycosylase activity of MBD4 mutants, which revealed the guanidinium moiety of Arg468 may play an important role in catalysis. D560N/R468K MBD4 bound to T:G mismatched DNA shows that the side chain amine moiety of the Lys stabilizes the flipped-out thymine by a water-mediated phosphate pinching, while the backbone carbonyl oxygen of the Lys engages in hydrogen bonds with N2 of the estranged guanine. Comparison of various DNA glycosylase structures implies the guanidinium and amine moieties of Arg and Lys, respectively, may involve in discriminating between substrate mismatches and nonsubstrate base pairs.

Apurinic/apyrimidinic endonuclease 1 (APE1) plays a critical role in the base excision repair (BER) pathway, which is responsible for the excision of apurinic sites (AP sites). In non-small cell lung cancer (NSCLC), APE1 is highly expressed and associated with poor patient prognosis. The suppression of APE1 could lead to the accumulation of unrepaired DNA damage in cells. Therefore, APE1 is viewed as an important marker of malignant tumors and could serve as a potent target for the development of antitumor drugs. In this study, we performed a high-throughput virtual screening of a small-molecule library using the three-dimensional structure of APE1 protein. Using the AP site cleavage assay and a cell survival assay, we identified a small molecular compound, NO.0449-0145, to act as an APE1 inhibitor. Treatment with NO.0449-0145 induced DNA damage, apoptosis, pyroptosis, and necroptosis in the NSCLC cell lines A549 and NCI-H460. This inhibitor was also able to impede cancer progression in an NCI-H460 mouse model. Moreover, NO.0449-0145 overcame both cisplatin- and erlotinib-resistance in NSCLC cell lines. These findings underscore the importance of APE1 as a therapeutic target in NSCLC and offer a paradigm for the development of small-molecule drugs that target key DNA repair proteins for the treatment of NSCLC and other cancers.

Targeting PARP1 [Poly(ADP-Ribose) Polymerase 1] for synthetic lethality is a new strategy for BRCA germ-line mutated or platinum sensitive ovarian cancers. However, not all patients respond due to intrinsic or acquired resistance to PARP1 inhibitor. Development of alternative synthetic lethality approaches is a high priority. DNA polymerase β (Polβ), a critical player in base excision repair (BER), interacts with PARP1 during DNA repair. Here we show that polβ deficiency is a predictor of platinum sensitivity in human ovarian tumours. Polβ depletion not only increased platinum sensitivity but also reduced invasion, migration and impaired EMT (epithelial to mesenchymal transition) of ovarian cancer cells. Polβ small molecular inhibitors (Pamoic acid and NSC666719) were selectively toxic to BRCA2 deficient cells and associated with double-strand breaks (DSB) accumulation, cell cycle arrest and increased apoptosis. Interestingly, PARG [Poly(ADP-Ribose) Glycohydrolase] inhibitor (PDD00017273) [but not PARP1 inhibitor (Olaparib)] was synthetically lethal in polβ deficient cells. Selective toxicity to PDD00017273 was associated with poly (ADP-ribose) accumulation, reduced nicotinamide adenine dinucleotide (NAD+) level, DSB accumulation, cell cycle arrest and increased apoptosis. In human tumours, polβ-PARG co-expression adversely impacted survival in patients. Our data provide evidence that polβ targeting is a novel strategy and warrants further pharmaceutical development in epithelial ovarian cancers.

Background: The most common oxidative DNA lesion is 8-oxoguanine (8-oxoG) which is mainly recognized and excised by the glycosylase OGG1, initiating the Base Excision Repair (BER) pathway. Telomeres are particularly sensitive to oxidative stress which disrupts telomere homeostasis triggering genome instability. Methods: We used U2OS OGG1-GFP osteosarcoma cell line to study the role of OGG1 at the telomeres in response to oxidative stress. Next, we investigated the effects of inactivating pharmacologically the BER during oxidative stress (OS) conditions by using a specific small molecule inhibitor of OGG1 (TH5487) in different human cell lines. Results: We have found that during OS, TH5487 effectively blocks BER initiation at telomeres causing accumulation of oxidized bases at this region, correlating with other phenotypes such as telomere losses, micronuclei formation and mild proliferation defects. Besides, the antimetabolite Methotrexate synergizes with TH5487 through induction of intracellular ROS formation, which potentiates TH5487 mediated telomere and genome instability in different cell lines. Conclusions: Our findings demonstrate that OGG1 is required to protect telomeres from OS and present OGG1 inhibitors as a tool to induce oxidative DNA damage at telomeres, with the potential for developing new combination therapies for cancer treatment.

Huntington’s disease (HD) is one of a large group of human disorders that are caused by expanded DNA repeats. These repeat expansion disorders can have repeat units of different size and sequence that can be located in any part of the gene and, while the pathological consequences of the expansion can differ widely, there is evidence to suggest that the underlying mutational mechanism may be similar. In the case of HD, the expanded repeat unit is a CAG trinucleotide located in exon 1 of the huntingtin (HTT) gene, resulting in an expanded polyglutamine tract in the huntingtin protein. Expansion results in neuronal cell death, particularly in the striatum. Emerging evidence suggests that somatic CAG expansion, specifically expansion occurring in the brain during the lifetime of an individual, contributes to an earlier disease onset and increased severity. In this review we will discuss mouse models of two non-CAG repeat expansion diseases, specifically the Fragile X-related disorders (FXDs) and Friedreich ataxia (FRDA). We will compare and contrast these models with mouse and patient-derived cell models of various other repeat expansion disorders and the relevance of these findings for somatic expansion in HD. We will also describe additional genetic factors and pathways that modify somatic expansion in the FXD mouse model for which no comparable data yet exists in HD mice or humans. These additional factors expand the potential druggable space for diseases like HD where somatic expansion is a significant contributor to disease impact.

This study assesses and compares the neurotoxic effects of proton and photon radiation on mitochondrial function and DNA repair capabilities of human astrocytes. Human astrocytes received either proton (0.5 Gy and 3 Gy), photon (0.5 Gy and 3 Gy), or sham-radiation treatment. The mRNA expression level of the DNA repair protein OGG1 was determined via RT-qPCR. The levels of 8-OHdG in the cell media were measured via ELISA. Real-time kinetic analysis of extracellular oxygen consumption rates was performed to assess mitochondrial function. Radiation-induced changes in mitochondrial mass and oxidative activity were assessed using fluorescent imaging with MitoTracker™ Green FM and MitoTracker™ Orange CM-H2TMRos dyes respectively. PCR was used to quantify the alteration in the mitochondrial DNA content, measured as the mitochondrial to nuclear DNA ratio. A significant increase in mitochondrial mass and levels of reactive oxygen species was observed after radiation treatment. Additionally, real-time PCR analysis indicated a significant depletion of mitochondrial DNA content in the irradiated cells when compared to the control. This was accompanied by a decreased gene expression of the DNA base-excision repair protein OGG1 and reduced clearance of 8-OHdG adducts from the genome. Photon radiation treatment was associated with a more detrimental cellular impact when compared to the same dose of proton radiation. These results are indicative of a radiation-induced dose-dependent decrease in mitochondrial function, an increase in senescence and astrogliosis, and impairment of the DNA repair capabilities in healthy glial cells. Photon irradiation was associated with a more significant disruption in mitochondrial function and base-excision repair mechanisms in vitro in comparison to proton treatment.

Chemotherapy plays an irreplaceable role in the treatment of GC, but currently available chemotherapeutic drugs are not ideal. The application of medicinal plants is an important direction for new drug discovery. Through drug screening of GC organoids, we determined that ailanthone has an anticancer effect on GC cells in vitro and in vivo. We also found that AIL can induce DNA damage and apoptosis in GC cells. Further transcriptome sequencing of PDX tissue indicated that AIL inhibited the expression of XRCC1, which plays an important role in DNA damage repair, and the results were also confirmed by western blotting. In addition, we found that AIL inhibited the expression of P23 and that inhibition of P23 decreased the expression of XRCC1, indicating that AIL can regulate XRCC1 via P23. The results of coimmunoprecipitation showed that AIL can inhibit the binding of P23 and XRCC1 to HSP90. These findings indicate that AIL can induce DNA damage and apoptosis in GC cells. Meanwhile, AIL can decrease XRCC1 activity by downregulating P23 expression to inhibit DNA damage repair. The present study sheds light on the potential application of new drugs isolated from natural medicinal plants for GC therapy.

Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1/Ref-1, also called APE1) is a multifunctional enzyme with crucial roles in DNA repair and reduction/oxidation (redox) signaling. APE1 was originally described as an endonuclease in the Base Excision Repair (BER) pathway. Further study revealed it to be a redox signaling hub regulating critical transcription factors (TFs). Although a significant amount of focus has been on the role of APE1 in cancer, recent findings support APE1 as a target in other indications, including ocular diseases [diabetic retinopathy (DR), diabetic macular edema (DME), and age-related macular degeneration (AMD)], inflammatory bowel disease (IBD) and others, where APE1 regulation of crucial TFs impacts important pathways in these diseases. The central responsibilities of APE1 in DNA repair and redox signaling make it an attractive therapeutic target for cancer and other diseases.

Objective This study describes the occurrence of a silent mutation in the RNA binding domain of nucleocapsid phosphoprotein (N protein) coding gene from SARS-CoV-2 that may consequence to a missense mutation by onset of another single nucleotide mutation. Results In the DNA sequence isolated from severe acute respiratory syndrome (SARS-CoV-2) in Iran, a coding sequence for the RNA binding domain of N protein was detected. The comparison of Chinese and Iranian DNA sequences displayed that a thymine (T) was mutated to cytosine (C), so “TTG” from China was changed to “CTG” in Iran. Both DNA sequences from Iran and China have been encoded for leucine. In addition, the second T in “CTG” in the DNA or uracil (U) in “CUG” in the RNA sequences from Iran can be mutated to another C by a missense mutation resulting from thymine DNA glycosylase (TDG) of human and base excision repair mechanism to produce “CCG” encoding for proline, which consequently may increase the affinity of the RNA binding domain of N protein to viral RNA and improve the transcription rate, pathogenicity, evasion from human immunity system, spreading in the human body, and risk of human-to-human transmission rate of SARS-CoV-2.

The six major mammalian DNA repair pathways were discovered as independent processes, each dedicated to remove specific types of lesions, but the past two decades have brought into focus the significant interplay between these pathways. In particular, several studies have demonstrated that certain proteins of the nucleotide excision repair (NER) and base excision repair (BER) pathways work in a cooperative manner in the removal of oxidative lesions. This review focuses on recent data showing how the NER proteins, XPA, XPC, XPG, CSA, CSB and UV-DDB, work to stimulate known glycosylases involved in the removal of certain forms of base damage resulting from oxidative processes, and also discusses how some oxidative lesions are probably directly repaired through NER. Finally, since many glycosylases are inhibited from working on damage in the context of chromatin, we detail how we believe UV-DDB may be the first responder in altering the structure of damage containing-nucleosomes, allowing access to BER enzymes.

Trinucleotide repeat (TNR) expansion and deletion are responsible for over 40 neurodegenerative diseases and associated with cancer. TNRs can undergo somatic instability that is mediated by DNA damage and repair and gene transcription. Recent studies have pointed toward a role for R-loops in causing TNR expansion and deletion, and it has been shown that base excision repair (BER) can result in CAG repeat deletion from R-loops in yeast. However, it remains unknown how BER in R-loops can mediate TNR instability. In this study, using biochemical approaches, we examined BER enzymatic activities and their influence on TNR R-loops. We found that AP endonuclease 1 incised an abasic site on the nontemplate strand of a TNR R-loop, creating a double-flap intermediate containing an RNA:DNA hybrid that subsequently inhibited polymerase β (pol β) synthesis of TNRs. This stimulated flap endonuclease 1 (FEN1) cleavage of TNRs engaged in an R-loop. Moreover, we showed that FEN1 also efficiently cleaved the RNA strand, facilitating pol β loop/hairpin bypass synthesis and the resolution of TNR R-loops through BER. Consequently, this resulted in fewer TNRs synthesized by pol β than those removed by FEN1, thereby leading to repeat deletion. Our results indicate that TNR R-loops preferentially lead to repeat deletion during BER by disrupting the balance between the addition and removal of TNRs. Our discoveries open a new avenue for the treatment and prevention of repeat expansion diseases and cancer.

This review summarizes the existing information concerning the genetic background of menopause and primary ovarian insufficiency (POI). There is overwhelming evidence that majority of genes are involved in double-strand break repair, mismatch repair, and base excision repair. The remaining loci were involved in cell energy metabolism and immune response. Gradual (or in case of rapid POI) accumulation of unrepaired DNA damage causes (premature) cell death and cellular senescence. This in turn leads to exhaustion of cell renewal capacity and cellular dysfunction in affected organs and eventually to aging of the entire soma. Similar erosion of the genome occurs within the germ cell line and the ovaries. Subsequently, the systemic “survival” response intentionally suppresses the sex-steroid hormonal output, which in turn may contribute to the onset of menopause. The latter occurs in particular when age-dependent DNA damage accumulation does not cease. Both effects are expected to synergize to promote (premature) ovarian silencing and install (early) menopause. Consequently, aging of the soma seems to be a primary driver for the loss of ovarian function in women. This challenges the current dogma which implies that loss of ovarian function initiates aging of the soma. It is time for a paradigm shift!

Significance The endogenous genome damage induced by mitochondrial/cytosolic reactive oxygen species (ROS) is well recognized. However, similar damage induced by nuclear ROS generated via histone/cytosine-phosphate-guanine (CpG) demethylation during transcription has not been scrupulously investigated. This report documents the formation of genomic oxidized bases and single-strand breaks during ligand-induced gene activation via histone/CpG demethylation. That repair of these damages occurs preferentially in promoters and is essential for transcriptional activation underscore the essentiality of promoter-specific repair for transcription. In contrast, heat shock (HS) induction generates double-strand breaks, the repair of which is essential for the activation of HS-responsive genes. This study thus implies gross underestimation of endogenous oxidative genome damage and highlights the intrinsic diversity of damage and distinct repair processes associated with transcription., Among several reversible epigenetic changes occurring during transcriptional activation, only demethylation of histones and cytosine-phosphate-guanines (CpGs) in gene promoters and other regulatory regions by specific demethylase(s) generates reactive oxygen species (ROS), which oxidize DNA and other cellular components. Here, we show induction of oxidized bases and single-strand breaks (SSBs), but not direct double-strand breaks (DSBs), in the genome during gene activation by ligands of the nuclear receptor superfamily. We observed that these damages were preferentially repaired in promoters via the base excision repair (BER)/single-strand break repair (SSBR) pathway. Interestingly, BER/SSBR inhibition suppressed gene activation. Constitutive association of demethylases with BER/SSBR proteins in multiprotein complexes underscores the coordination of histone/DNA demethylation and genome repair during gene activation. However, ligand-independent transcriptional activation occurring during heat shock (HS) induction is associated with the generation of DSBs, the repair of which is likewise essential for the activation of HS-responsive genes. These observations suggest that the repair of distinct damages induced during diverse transcriptional activation is a universal prerequisite for transcription initiation. Because of limited investigation of demethylation-induced genome damage during transcription, this study suggests that the extent of oxidative genome damage resulting from various cellular processes is substantially underestimated.

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA.

DNA damage may develop at any dose of ionizing radiation. DNA damage activates pathways that regulate cell growth and division or coordinate its replication and repair. The repair pathways, base excision repair (BER) and single-strand break repair (SSBR), can repair such damages efficiently and maintain genome integrity. Loss of this repair process or alteration of its control will be associated with serious outcomes for cells and individuals. This study aimed to determine the relationship between XRCC1 (Arg194Trp, Arg280His, and Arg399Gln), OGG1 (Ser326Cys), and XRCC3 (Thr241Met) SNPs and DNA damage and to identify high-risk individuals with reduced DNA repair capacity. This case-control study was conducted on 80 subjects; 50 subjects working in Clinical Oncology and Nuclear Medicine Department in Assiut University Hospital along with 30 controls. A total of 1 mL blood samples were collected for Single-Cell Gel Electrophoresis Technique (Comet Assay) for detection of DNA damage in those subjects. A total of 3 mL fresh blood samples were collected and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)–based technique. DNA damage detected by comet test was significantly high in IR-exposed workers than control. Statistically high significant difference was found in exposed subjects versus control subjects regarding the frequencies of the variant alleles of hOGG1326, XRCC1280 & 399, and XRCC3241. The level of DNA damage was not affected by OGG1326 SNPs when comparing subjects of wild genotype with those of (pooled) variants either in the exposed staff or in the control group while XRCC1280, 399 and XRCC3241 variant alleles had an influence on the studied DNA damage biomarker. Moreover, genotyping distribution pattern was highly variable in relation to gender. The present study indicated a relationship between DNA damage detected by comet test and single nucleotide polymorphisms in genes coding for DNA certain repair enzymes. Individuals occupationally exposed to low doses of ionizing radiation could be at great risk and more susceptible to the increased DNA damage if they have inherited genetic polymorphism.

MutY glycosylase excises adenines misincorporated opposite the oxidatively damaged lesion, 8-oxo-7,8-dihydroguanine (OG), to initiate base excision repair and prevent G to T transversion mutations. Successful repair requires MutY recognition of the OG:A mispair amidst highly abundant and structurally similar undamaged DNA base pairs. Herein we use a combination of in vitro and bacterial cell repair assays with single-molecule fluorescence microscopy to demonstrate that both a C-terminal domain histidine residue and the 2-amino group of OG base are critical for MutY detection of OG:A sites. These studies are the first to directly link deficiencies in MutY lesion detection with incomplete cellular repair. These results suggest that defects in lesion detection of human MutY (MUTYH) variants may prove predictive of early-onset colorectal cancer known an MUTYH-associated polyposis. Furthermore, unveiling these specific molecular determinants for repair makes it possible to envision new MUTYH-specific cancer therapies.

Poly ADP-ribose polymerase (PARP) enzymes are critical to base excision repair of single-stranded DNA breaks. PARP inhibitors (PARPi) have been shown to induce synthetic lethality in some patients ...

Eukaryotic DNA polymerase β (Pol β) plays an important role in cellular DNA repair, as it fills short gaps in dsDNA that result from removal of damaged bases. Since defects in DNA repair may lead to cancer and genetic instabilities, Pol β has been extensively studied, especially its mechanisms for substrate binding and a fidelity-related conformational change referred to as "fingers closing." Here, we applied single-molecule FRET to measure distance changes associated with DNA binding and prechemistry fingers movement of human Pol β. First, using a doubly labeled DNA construct, we show that Pol β bends the gapped DNA substrate less than indicated by previously reported crystal structures. Second, using acceptor-labeled Pol β and donor-labeled DNA, we visualized dynamic fingers closing in single Pol β-DNA complexes upon addition of complementary nucleotides and derived rates of conformational changes. We further found that, while incorrect nucleotides are quickly rejected, they nonetheless stabilize the polymerase-DNA complex, suggesting that Pol β, when bound to a lesion, has a strong commitment to nucleotide incorporation and thus repair. In summary, the observation and quantification of fingers movement in human Pol β reported here provide new insights into the delicate mechanisms of prechemistry nucleotide selection.

Objective: X-ray cross-complementing group 1 (XRCC1) and 8 Oxo guanine DNA-glycosylase 1 (OGG1) genes are implicated in the repair of single-stranded breaks (SSBRs) and base excision repair (BER) pathways. Common polymorphisms in DNA repair genes are supposed to decrease the capability of DNA repair and cause genetic instability. This study was designed to investigate the association between XRCC1 (rs25487) and OGG1 (rs1052133) polymorphisms and susceptibility to colorectal cancer (CRC) in the Ahvaz city, south-west Iran. Methods: This case- control study comprised 150 patients and 150 controls that were selected from 2 educational hospitals in Ahvaz. They were matched for age and gender, and their genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results: Our results indicate that the frequency of the Gln (A) allele of XRCC1 (rs25487) is significantly higher in colorectal cancer patients, compare to controls (p = 0.01, OR: 1.54, 95% CI 1.9–13.3). Significant increased risk of cancer was observed in XRCC1 (rs25487) genotypes (p = 0.001 OR: 5.3, 95% CI 1.9–14.2 for Gln / Gln), while no association was found between OGG1 (rs1052133) and colorectal cancer risk (p = 0.6). Conclusion: Our study suggests that XRCC1 (rs25487) polymorphism might be associated with an increasing risk of CRC in Ahvaz. It also demonstrates positive correlation between the XRCC1 (rs25487) genotypes and demographic characteristics, such as smoking and increased age in patients and control groups.

Long-term arsenic exposure is a worldwide public health problem that causes serious harm to human health. The liver is the main target organ of arsenic toxicity; arsenic induces disruption of the DNA damage repair pathway, but its mechanisms remain unclear. In recent years, studies have found that epigenetic mechanisms play an important role in arsenic-induced lesions. In this study, we conducted experiments in vitro using normal human liver cells (L-02) to explore the mechanism by which the histone demethylase JHDM2A regulates H3K9 dimethylation (me2) in response to arsenic-induced DNA damage. Our results indicated that arsenic exposure upregulated the expression of JHDM2A, downregulated global H3K9me2 modification levels, increased the H3K9me2 levels at the promoters of base excision repair (BER) genes (N-methylpurine-DNA glycosylase [MPG], XRCC1 and poly(ADP-ribose)polymerase 1) and inhibited their expression levels, causing DNA damage in cells. In addition, we studied the effects of overexpression and inhibition of JHDM2A and found that JHDM2A can participate in the molecular mechanism of arsenic-induced DNA damage via the BER pathway, which may not be involved in the BER process because H3K9me2 levels at the promoter region of the BER genes were unchanged following JHDM2A interference. These results suggest a potential mechanism by which JHDM2A can regulate the MPG and XRCC1 genes in the process of responding to DNA damage induced by arsenic exposure and can participate in the process of DNA damage repair, which provides a scientific basis for understanding the epigenetic mechanisms and treatments for endemic arsenic poisoning.

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1.

Chromatin-associated architectural proteins are part of a fundamental support system for cellular DNA-dependent processes and can maintain/modulate the efficiency of DNA replication, transcription, and DNA repair. Interestingly, prognostic outcomes of many cancer types have been linked with the expression levels of several of these architectural proteins. The high mobility group box (HMGB) architectural protein family has been well studied in this regard. The differential expression levels of HMGB proteins and/or mRNAs and their implications in cancer etiology and prognosis present the potential of novel targets that can be explored to increase the efficacy of existing cancer therapies. HMGB1, the most studied member of the HMGB protein family, has pleiotropic roles in cells including an association with nucleotide excision repair, base excision repair, mismatch repair, and DNA double-strand break repair. Moreover, the HMGB proteins have been identified in regulating DNA damage responses and cell survival following treatment with DNA-damaging agents and, as such, may play roles in modulating the efficacy of chemotherapeutic drugs by modulating DNA repair pathways. Here, we discuss the functions of HMGB proteins in DNA damage processing and their potential roles in cancer etiology, prognosis, and therapeutics.

The presence of cancer stem cells (CSCs) in the tumor microenvironment is responsible for the development of chemoresistance and recurrence of cancer. Our previous investigation revealed the anticancer mechanism of quinacrine-based silver and gold hybrid nanoparticles (QAgNP and QAuNP) in oral cancer cells, but to avoid cancer recurrence, it is important to study the effect of these nanoparticles (NPs) on CSCs. Here, we developed an in vitro CSCs model using SCC-9 oral cancer cells and validated via FACS analysis. Then, 40-60% of cells were found to be CD44+/CD133+ and CD24-. QAuNP showed excellent anti-CSC growth potential against SCC-9-cancer stem like cells (IC50 = 0.4 μg/mL) with the down-regulation of representative CSC markers. Prolonged exposure of QAuNP induced the S-phase arrest and caused re-replication shown by the extended G2/M population and apoptosis to SCC-9-CSC like cells. Up-regulation of BAX, PARP cleavage, and simultaneous down-regulation of Bcl-xL in prolonged treatment to CSCs suggested that the majority of the cells have undergone apoptosis. QAuNP treatment also caused a loss in DNA repair in CSCs. Mostly, the base excision repair (BER) components (Fen-1, DNA ligase-1, Pol-β, RPA, etc.) were significantly down-regulated after QAuNP treatment, which suggested its action against DNA repair machinery. The replication fork maintenance-related proteins, RAD 51 and BRCA-2, were also deregulated. Very surprisingly, depletion of WRN (an interacting partner for Pre-RC and Fen-1) and a significant increase in expression of fork-degrading nuclease MRE-11 in 96 h treated NPs were observed. Results suggest QAuNP treatment caused excessive DNA damage and re-replication mediated replication stress (RS) and stalling of the replication fork. Inhibition of BER components hinders the flap clearance activity of Fen-1, and it further caused RS and stopped DNA synthesis. Overall, QAuNP treatment led to irreparable replication fork movement, and the stalled replication fork might have degraded by MRE-11, which ultimately results in apoptosis and the death of the CSCs.

A plethora of molecular strategies are employed by breast cancer stem cells (bCSCs) to evade chemotherapy-induced death signals, redox modulation being a crucial factor among those. Here, we observed that bCSCs are resistant to DNA damage and generate low ROS upon doxorubicin (Dox) treatment. Further exploration revealed inherently high NEIL2, a base excision repair (BER) enzyme that plays a key regulatory role in repairing DNA damage, in bCSCs. However, its role in modulating the redox status of bCSCs remains unexplored. In addition, Dox not only upregulates NEIL2 in bCSCs at both transcriptional and translational levels but also declines p300-induced acetylation thus activating NEIL2 and providing a protective effect against the stress inflicted by the genotoxic drug. However, when the redox status of bCSCs is altered by inducing high ROS, apoptosis of the resistant population is accomplished. Subsequently, when NEIL2 is suppressed in bCSCs, chemo-sensitization of the resistant population is enabled by redox reconditioning via impaired DNA repair. This signifies a possibility of therapeutically disrupting the redox balance in bCSCs to enhance their chemo-responsiveness. Our search for an inhibitor of NEIL2 revealed that vitamin B6, i.e., pyridoxine (PN), hinders NEIL2-mediated transcription-coupled repair process by not only decreasing NEIL2 expression but also inhibiting its association with RNA Pol II, thus stimulating DNA damage and triggering ROS. As a consequence of altered redox regulation, bCSCs become susceptible towards Dox, which then induces apoptosis via caspase cascade. These findings signify that PN enhances chemo-responsiveness of bCSCs via redox reconditioning.