X-ray Repair Cross-complementing Protein Research Articles

Single nucleotide polymorphisms rs148582811 regulates its host gene ARVCF expression to affect nicotine-associated hippocampus-dependent memory

Although numerous susceptibility loci are nominated for nicotine dependence (ND), no report showed any association of ARVCF with ND. Through genome-wide sequencing analysis, we first identified genetic variants associated nominally with ND and then replicated them in an independent sample. Of the six replicated variants, rs148582811 in ARVCF located in the enhancer-associated marker peak is attractive. The effective-median-based Mendelian randomization analysis indicated that ARVCF is a causal gene for ND. RNA-seq analysis detected decreased ARVCF expression in smokers compared to nonsmokers. Luciferase reporter assays indicated that rs148582811 and its located DNA fragment allele-specifically regulated ARVCF expression. Immunoprecipitation analysis revealed that transcription factor X-ray repair cross-complementing protein 5 (XRCC5) bound to the DNA fragment containing rs148582811 and allele-specifically regulated ARVCF expression at the mRNA and protein levels. With the Arvcf knockout mouse model, we showed that Arvcf deletion not only impairs hippocampus-dependent learning and memory, but also alleviated nicotine-induced memory deficits.

Regulation of base excision repair during adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells

Bone marrow-derived human mesenchymal stem cells (hMSCs) can differentiate into various lineages, such as chondrocytes, adipocytes, osteoblasts, and neuronal lineages. It has been shown that the high-efficiency DNA-repair capacity of hMSCs is decreased during their differentiation. However, the underlying its mechanism during adipogenesis and osteogenesis is unknown. Herein, we investigated how alkyl-damage repair is modulated during adipogenic and osteogenic differentiation, especially focusing on the base excision repair (BER) pathway. Response to an alkylation agent was assessed via quantification of the double-strand break (DSB) foci and activities of BER-related enzymes during differentiation in hMSCs. Adipocytes showed high resistance against methyl methanesulfonate (MMS)-induced alkyl damage, whereas osteoblasts were more sensitive than hMSCs. During the differentiation, activities, and protein levels of uracil-DNA glycosylase were found to be regulated. In addition, ligation-related proteins, such as X-ray repair cross-complementing protein 1 (XRCC1) and DNA polymerase β, were upregulated in adipocytes, whereas their levels and recruitment declined during osteogenesis. These modulations of BER enzyme activity during differentiation influenced DNA repair efficiency and the accumulation of DSBs as repair intermediates in the nucleus. Taken together, we suggest that BER enzymatic activity is regulated in adipogenic and osteogenic differentiation and these alterations in the BER pathway led to different responses to alkyl damage from those in hMSCs.

PAXX binding to the NHEJ machinery explains functional redundancy with XLF.

Nonhomologous end joining is a critical mechanism that repairs DNA double-strand breaks in human cells. In this work, we address the structural and functional role of the accessory protein PAXX [paralog of x-ray repair cross-complementing protein 4 (XRCC4) and XRCC4-like factor (XLF)] in this mechanism. Here, we report high-resolution cryo-electron microscopy (cryo-EM) and x-ray crystallography structures of the PAXX C-terminal Ku-binding motif bound to Ku70/80 and cryo-EM structures of PAXX bound to two alternate DNA-dependent protein kinase (DNA-PK) end-bridging dimers, mediated by either Ku80 or XLF. We identify residues critical for the Ku70/PAXX interaction in vitro and in cells. We demonstrate that PAXX and XLF can bind simultaneously to the Ku heterodimer and act as structural bridges in alternate forms of DNA-PK dimers. Last, we show that engagement of both proteins provides a complementary advantage for DNA end synapsis and end joining in cells.

TEAD Proteins Associate With DNA Repair Proteins to Facilitate Cellular Recovery From DNA Damage

Transcriptional enhanced associate domain family members 1 to 4 (TEADs) are a family of four transcription factors and the major transcriptional effectors of the Hippo pathway. In order to activate transcription, TEADs rely on interactions with other proteins, such as the transcriptional effectors Yes-associated protein and transcriptional co-activator with PDZ-binding motif. Nuclear protein interactions involving TEADs influence the transcriptional regulation of genes involved in cell growth, tissue homeostasis, and tumorigenesis. Clearly, protein interactions for TEADs are functionally important, but the full repertoire of TEAD interaction partners remains unknown. Here, we employed an affinity purification mass spectrometry approach to identify nuclear interacting partners of TEADs. We performed affinity purification mass spectrometry experiment in parallel in two different cell types and compared a wildtype TEAD bait protein to a nuclear localization sequence mutant that does not localize to the nucleus. We quantified the results using SAINT analysis and found a significant enrichment of proteins linked to DNA damage including X-ray repair cross-complementing protein 5 (XRCC5), X-ray repair cross-complementing protein 6 (XRCC6), poly(ADP-ribose) polymerase 1 (PARP1), and Rap1-interacting factor 1 (RIF1). In cellular assays, we found that TEADs co-localize with DNA damage–induced nuclear foci marked by histone H2AX phosphorylated on S139 (γH2AX) and Rap1-interacting factor 1. We also found that depletion of TEAD proteins makes cells more susceptible to DNA damage by various agents and that depletion of TEADs promotes genomic instability. Additionally, depleting TEADs dampens the efficiency of DNA double-stranded break repair in reporter assays. Our results connect TEADs to DNA damage response processes, positioning DNA damage as an important avenue for further research of TEAD proteins.

PS-BPB02-2: POLYMORPHISM OF DNA REPAIR GENE IS ASSOCIATED WITH HYPERTENSION-INDUCED LEFT VENTRICULAR HYPERTROPHY - ROLE OF DNA DAMAGE AND CELL SENESCENCE

Objective: Deficiency of X-ray repair cross-complementing protein 3 (XRCC3), a DNA-damage repair molecule, and 241Met variant of XRCC3 are prone to increase endoreduplication, resulting in pathological polyploidy. In this study we investigated the impact of XRCC3 polymorphism on the incidence of hypertension-induced left ventricular hypertrophy (LVH) and underlying mechanisms. Design and method: Patients under hemodialysis (n = 77) were genotyped for XRCC3 Thr241Met polymorphism. LVH was determined by echocardiography. To investigate the possible mechanisms for LVH, human XRCC3 cDNA of 241Thr or that of 241Met was introduced into cultured cells. The surface area of the cells was measured and spontaneous DNA double-strand breaks were detected by immunofluorescent study with anti-53BP1 antibody. The mRNA levels of basal and TNF-α-stimulated MCP-1 were quantified by real-time RT PCR. FACS analysis was performed to count the percentage of polyploidy. Results: In patients under hemodialysis, XRCC3 241Thr/Met genotype was more frequent in LVH (+) group than in LVH (-) group (42.3% vs. 13.7%, χ2 = 7.85, p = 0.0051). To investigate the possible mechanisms for LVH, following in vitro experiments were performed. The surface area of Chinese hamster ovary cells expressing XRCC3 241Met variant was larger than those expressing 241Thr. The accumulation of spontaneous DNA double-strand breaks (DSBs) was increased in NIH3T3 cells expressing 241Met (3T3–241Met) than those expressing 241Thr (3T3–241Thr). Ionizing radiation-induced cell senescence was significantly enhanced in 3T3–241Met than in 3T3–241Thr. The mRNA levels of basal and TNF-α-stimulated MCP-1 were higher in 3T3–241Met variant than 3T3–241Thr. Finally, FACS analysis revealed that the cell percentage in G2/M phase including polyploidy was significantly increased in 3T3–241Met compared with 3T3–241Thr. In addition, the basal mRNA level of MCP-1 was positively correlated with the cell percentage in G2/M phase and polyploidy. Conclusion: The current study suggests that XRCC3 241Met variant increases the risk of LVH via the accumulation of DNA damage, alteration of cell cycle progression, and induction of cell senescence and proinflammatory phenotype.

Influence of differential arsenic exposure on cellular redox homeostasis of exposed rural women of West Bengal.

The metalloid arsenic (As) induces oxidative stress is a well-known fact. However, the extent of variation of oxidative stress according to different exposure levels of As in groundwater and the mechanism responsible for As mediated oxidative stress is yet to be elucidated in a human population of West Bengal. In the present study, we have investigated the impact of low level (> 10 ≤ 50µg/L) and high-level groundwater As (> 50µg/L) on cellular redox status, DNA damage, and repair mechanisms in chronically exposed rural women of West Bengal. Prediction models of ordinary least square regression of nail As, forced vital capacity (FVC) %, and that of forced expiratory volume during the first one second (FEV1) % deciphered that accumulation of As in nails may predict hemoglobin deficiency. Moreover, consumption of As-laced water tends to decrease FEV1% and FVC%. A strong positive correlation was observed between water and nail As level and reactive oxygen species (ROS) generation. ROS, perturbed nuclear factor erythroid 2-related factor 2(Nrf2)/ Kelch-like ECH-associated protein 1 (Keap1) redox regulation, compromised antioxidant defense machinery-superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione transferase (GST), induced DNA damage, and suppressed DNA repair proteins-poly ADP ribose polymerase1(PARP1)/ X-ray repair cross-complementing protein 1(XRCC1)/ 8-oxoguanine glycosylase (OGG1) in a dose-dependent manner. All the low and high As areas had very high cancer risk values for the exposed population. It has been predicted that if the As level in the drinking water of a 40-year adult increases by 2 ug/L, the likelihood of the cancer risk will increase by 10%, keeping the body weight and amount of water intake constant. Thus, long-term exposure to either low or high As is seriously affecting the lives of asymptomatic women who are vulnerable to developing carcinogenic changes after a period of latency.

Serum levels of vitamin A, selenium, and better dietary total antioxidant capacity are related to lower oxidative DNA damage: A cross-sectional study of individuals at cardiovascular risk

The risk for cardiovascular diseases (CVR) has been associated with oxidative DNA damage, but the genetic and environmental factors involved in the antioxidant and DNA repair system contributing to this damage are unknown. The aim was to evaluate the levels of oxidative DNA damage in CVR subjects and how it is related with some genetic and nutritional factors. The cross-sectional study evaluated 136 individuals of both sexes, aged 20–59 years, with at least one cardiovascular risk factor. The global risk score was used to classify individuals at low, intermediate, and high cardiovascular risk. The dietary total antioxidant capacity (DTAC) was calculated using table with FRAP values. The oxidative DNA damage was verified by the comet assay. The variants null of Glutathione-S-transferases Mu1 and Theta 1(GSTM1 and GSTT1) and rs25487 of X-Ray Repair Cross Complementing Protein 1 (XRCC1) were analyzed by real-time PCR and PCR-RFLP, respectively. The oxidative DNA damage was higher in patients with intermediate/high CVR than in patients with low CVR (P=.01). Individuals with GSTT1/GSTM1 null genotypes or arg/gln+gln/gln genotypes of the XRCC1 (rs25487) gene showed similar levels of oxidative DNA damage compared wild genotype. Multivariate regression analysis demonstrated that oxidative DNA damage in individuals with CVR depends on serum levels of vitamin A, selenium, and DTAC independently of the other factors [F(6.110)=8.213; P<.001; R2=0.330]. These findings suggest that nutritional factors such as DTAC, vitamin A and selenium may have a protective effect against oxidative DNA damage in these individuals.

Role of PPAR-γ in diabetes-induced testicular dysfunction, oxidative DNA damage and repair in leptin receptor-deficient obese type 2 diabetic mice

The testis expresses peroxisome proliferator-activated receptor-γ (PPAR-γ), but its involvement in regulating diabetes-induced testicular dysfunction and DNA damage repair is not known. Pioglitazone-induced activation of PPAR-γ for 12 weeks in db/db obese diabetic mice increases bodyweights and reduces blood glucose levels, but PPAR-γ inhibition by 2-chloro-5-nitro-N-phenylbenzamide does not alter these parameters; instead, improves testis and epididymis weights and sperm count. Neither activation nor inhibition of PPAR-γ normalizes the diabetes-induced seminiferous epithelial degeneration. The PPAR-γ activation normalizes testicular lipid peroxidation, but its inhibition reduces lipid peroxidation and oxidative DNA damage (8-oxo-dG) in diabetic mice. As a response to diabetes-induced oxidative DNA damage, the base-excision repair (BER) mechanism proteins- 8-oxoguanine DNA glycosylases (OGG1/2) and X-ray repair cross-complementing protein-1 (XRCC1) increase, whereas the redox-factor-1 (REF1), DNA polymerase (pol) δ and poly (ADP-ribose) polymerase-1 (PARP1) show a tendency to increase suggesting an attempt to repair the oxidative DNA damage. The PPAR-γ stimulation inhibits OGG2, DNA pol δ, and XRCC1 in diabetic mice testes, but PPAR-γ inhibition reduces oxidative DNA damage and normalizes BER protein levels. In conclusion, type 2 diabetes negatively affects testicular structure and function and increases oxidative DNA damage and BER protein levels due to increased DNA damage. The PPAR-γ modulation does not significantly affect the structural changes in the testis. The PPAR-γ stimulation aggravates diabetes-induced effects on testis, including oxidative DNA damage and BER proteins, but PPAR-γ inhibition marginally recovers these diabetic effects indicating the involvement of the receptor in the reproductive effects of diabetes.

Role of Paralogue of XRCC4 and XLF in DNA Damage Repair and Cancer Development.

Non-homologous end joining (cNHEJ) is a major pathway to repair double-strand breaks (DSBs) in DNA. Several core cNHEJ are involved in the progress of the repair such as KU70 and 80, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Artemis, X-ray repair cross-complementing protein 4 (XRCC4), DNA ligase IV, and XRCC4-like factor (XLF). Recent studies have added a number of new proteins during cNHEJ. One of the newly identified proteins is Paralogue of XRCC4 and XLF (PAXX), which acts as a scaffold that is required to stabilize the KU70/80 heterodimer at DSBs sites and promotes the assembly and/or stability of the cNHEJ machinery. PAXX plays an essential role in lymphocyte development in XLF-deficient background, while XLF/PAXX double-deficient mouse embryo died before birth. Emerging evidence also shows a connection between the expression levels of PAXX and cancer development in human patients, indicating a prognosis role of the protein. This review will summarize and discuss the function of PAXX in DSBs repair and its potential role in cancer development.

A Prognostic Model Based on XRCC1 Gene Polymorphisms and Clinicopathological Factors to Predict One-Year Survival of Advanced Epithelial Ovarian Cancer Patients

Prognosis of advanced stage epithelial ovarian cancer (EOC) patients remains poor due to recurrence and disease progression after surgery and chemotherapy. Polymorphism in the genes involved in DNA repair, i.e. x-ray repair cross complementing protein-1 (XRCC1) and XRCC3 could alter function, diminish repair kinetics and may play a role in tumor resistance to therapy. This study was aimed to predict the prognosis of advanced stage EOC patients based on the polymorphism of XRCC1 and XRCC3 genes and the clinicopathology. This was an ambispective cohort study in EOC patients treated in Fatmawati General Central Hospital and its network hospitals in Jakarta between 2011 and 2016. Demographic data and clinicopathology was obtained from the medical record. Polymorphisms of XRCC1 and XRCC3 genes were detected by using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Survival analyses were performed using the Kaplan-Meier curve and Cox proportional hazard test. Multivariate analyses were performed to obtain prediction model for 1-year survival. Scoring system was developed and tested using the receiver operating characteristic (ROC) curve analyses. A total of 129 eligible cases were included. Patients’ mean age was 49.6 years. Most patients (93.8%) were having stage III disease. Mortality at 1-year was associated with three independent variables, i.e. histopathology subtype, residual tumor &gt; 1 cm, and XRCC1 gene Arg399Gln polymorphisms (Arg/Gln or Gln/Gln alleles). These prediction models gave 85.3% accuracy by ROC curve analysis and 82.7% posttest probability. Prognosis of advanced stage EOC patients can be predicted from several clinicopathological factors and XRCC1 gene polymorphisms. Validation study is needed to confirm these findings.

Gene-environment interactions related to maternal exposure to environmental and lifestyle-related chemicals during pregnancy and the resulting adverse fetal growth: a review.

BackgroundThere are only limited numbers of reviews on the association of maternal-child genetic polymorphisms and environmental and lifestyle-related chemical exposure during pregnancy with adverse fetal growth. Thus, this article aims to review: (1) the effect of associations between the above highlighted factors on adverse fetal growth and (2) recent birth cohort studies regarding environmental health risks.MethodsBased on a search of the PubMed database through August 2021, 68 epidemiological studies on gene-environment interactions, focusing on the association between environmental and lifestyle-related chemical exposure and adverse fetal growth was identified. Moreover, we also reviewed recent worldwide birth cohort studies regarding environmental health risks.ResultsThirty studies examined gene-smoking associations with adverse fetal growth. Sixteen maternal genes significantly modified the association between maternal smoking and adverse fetal growth. Two genes significantly related with this association were detected in infants. Moreover, the maternal genes that significantly interacted with maternal smoking during pregnancy were cytochrome P450 1A1 (CYP1A1), X-ray repair cross-complementing protein 3 (XRCC3), interleukin 6 (IL6), interleukin 1 beta (IL1B), human leukocyte antigen (HLA) DQ alpha 1 (HLA-DQA1), HLA DQ beta 1 (HLA-DQB1), and nicotinic acetylcholine receptor. Fetal genes that had significant interactions with maternal smoking during pregnancy were glutathione S-transferase theta 1 (GSTT1) and fat mass and obesity-associated protein (FTO). Thirty-eight studies examined the association between chemical exposures and adverse fetal growth. In 62 of the 68 epidemiological studies (91.2%), a significant association was found with adverse fetal growth. Across the studies, there was a wide variation in the analytical methods used, especially with respect to the genetic polymorphisms of interest, environmental and lifestyle-related chemicals examined, and the study design used to estimate the gene-environment interactions. It was also found that a consistently increasing number of European and worldwide large-scale birth cohort studies on environmental health risks have been conducted since approximately 1996.ConclusionThere is some evidence to suggest the importance of gene-environment interactions on adverse fetal growth. The current knowledge on gene-environment interactions will help guide future studies on the combined effects of maternal-child genetic polymorphisms and exposure to environmental and lifestyle-related chemicals during pregnancy.Supplementary informationThe online version contains supplementary material available at https://doi.org/10.1265/ehpm.21-00033.

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals Myrocin G as an Inhibitor of the Nonhomologous End Joining DNA Repair Pathway.

Chemoproteomic profiling of cysteines has emerged as a powerful method for screening the proteome-wide targets of cysteine-reactive fragments, drugs, and natural products. Herein, we report the development and an in-depth evaluation of a tetrafluoroalkyl benziodoxole (TFBX) as a cysteine-selective chemoproteomic probe. We show that this probe features numerous key improvements compared to the traditionally used cysteine-reactive probes, including a superior target occupancy, faster labeling kinetics, and broader proteomic coverage, thus enabling profiling of cysteines directly in live cells. In addition, the fluorine "signature" of probe 7 constitutes an additional advantage resulting in a more confident adduct-amino acid site assignment in mass-spectrometry-based identification workflows. We demonstrate the utility of our new probe for proteome-wide target profiling by identifying the cellular targets of (-)-myrocin G, an antiproliferative fungal natural product with a to-date unknown mechanism of action. We show that this natural product and a simplified analogue target the X-ray repair cross-complementing protein 5 (XRCC5), an ATP-dependent DNA helicase that primes DNA repair machinery for nonhomologous end joining (NHEJ) upon DNA double-strand breaks, making them the first reported inhibitors of this biomedically highly important protein. We further demonstrate that myrocins disrupt the interaction of XRCC5 with DNA leading to sensitization of cancer cells to the chemotherapeutic agent etoposide as well as UV-light-induced DNA damage. Altogether, our next-generation cysteine-reactive probe enables broader and deeper profiling of the cysteinome, rendering it a highly attractive tool for elucidation of targets of electrophilic small molecules.

X-ray repair cross-complementing protein 1 (XRCC1) loss promotes \u03b2-lapachone \u2013induced apoptosis in pancreatic cancer cells

Backgroundβ-lapachone (β-lap), the NQO1 bioactivatable drug, is thought to be a promising anticancer agent. However, the toxic side effects of β-lap limit the drug use, highlighting the need for a thorough understanding of β-lap’s mechanism of action. β-lap undergoes NQO1-dependent futile redox cycling, generating massive ROS and oxidative DNA lesions, leading to cell death. Thus, base excision repair (BER) pathway is an important resistance factor. XRCC1, a scaffolding component, plays a critical role in BER.MethodsWe knocked down XRCC1 expression by using pLVX-shXRCC1 in the MiaPaCa2 cells and BxPC3 cells and evaluated β-lap-induced DNA lesions by γH2AX foci formation and alkaline comet assay. The cell death induced by XRCC1 knockdown + β-lap treatment was analysed by relative survival, flow cytometry and Western blotting analysis.ResultsWe found that knockdown of XRCC1 significantly increased β-lap-induced DNA double-strand breaks, comet tail lengths and cell death in PDA cells. Furthermore, we observed combining XRCC1 knockdown with β-lap treatment switched programmed necrosis with β-lap monotherapy to caspase-dependent apoptosis.ConclusionsThese results indicate that XRCC1 is involved in the repair of β-lap-induced DNA damage, and XRCC1 loss amplifies sensitivity to β-lap, suggesting targeting key components in BER pathways may have the potential to expand use and efficacy of β-lap for gene-based therapy.

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells.

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (1O2) only at telomeres or mitochondria in Jurkat T cells. We found that targeted 1O2 production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted 1O2 formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet 1O2 formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.

Loss of the Base Excision Repair Gene Apex1 Leads to Dysfunctional Adult Hematopoietic Stem and Progenitor Cells

Postnatal hematopoietic stem (and progenitor) cells (HS(P)Cs) are especially vulnerable to oxidative stress, leading to early hematopoietic senescence and/or malignant transformation. Elevated intracellular reactive oxygen species (ROS) can, among others, oxidize nucleotides, and thus can result in genotoxicity and mutagenesis if left unrepaired. Oxidized bases, as well as other spontaneous single base modifications, are recognized and repaired by the base excision repair (BER) pathway. Hence, the BER pathway is crucial to maintain genome integrity. In contrast to other DNA repair pathways however, the role of BER in maintaining HSPC functionality remains enigmatic, chiefly because knockout (KO) of BER genes is in many cases embryonic lethal.BER is a complex multi-step repair process. After initial removal and excision of the damaged base, the apurinic/apyrimidinic (AP) site is processed by the AP endonuclease (APEX1) enzyme. At this point, the BER pathway branches into 2 sub-pathways, namely the short-patch (SP-BER; wherein DNA polymerase beta (Polβ), Ligase III (Lig3) together with X-ray repair cross-complementing protein 1 (Xrcc1) are active) and the long-patch BER (LP-BER; wherein Lig1, Flap Structure-Specific Endonuclease 1 (Fen1), and sometimes Polβ are active) for the repair synthesis and the gap filling steps.In this study we wished to address the role of BER in adult hematopoiesis. Therefore, we used CRISPR-Cas9 to KO different BER genes in adult bone marrow (BM) HS(P)Cs, including two genes common to the BER (sub-)pathway(s) (Apex1 and Polβ) as well as one gene in the SP-BER (Xrcc1) and one gene in the LP-BER (Lig1) pathway. The effect thereof was evaluated on HS(P)C repopulation in vivo as well as on HS(P)C expansion during long-term in vitro culture (using the culture medium described by Wilkinson et al., Nature 2019). All CRISPR-Cas9 experiments were validated using a second sgRNA targeting the selected BER genes.Lig1-KO caused in vivo HSPC dysfunction: at 20 weeks post-transplantation, significantly less Lig1 KO cells were observed in the committed progenitor (HPC) and lineage committed (Lin +) BM compartments. By contrast, KO of Xrcc1 had only minor effects on HS(P)C repopulation, but we observed increased HSC expansion and myeloid biased differentiation in some recipient mice, which might correspond to clonal hematopoiesis and is consistent with the finding of XRCC1 loss-of-function mutation in myelodysplastic patients (Joshi et al, Ann Hematol 2016). Knockout of Polβ did not affect hematopoiesis in vivo or in vitro. The most severe phenotype was observed when we knocked out Apex1, as Apex1-KO HS(P)Cs failed to repopulate irradiated recipient mice. Already after 2 weeks, significantly less Apex1 deficient cells were detected in the different blood lineages and nearly no CRISPR-Cas9 KO cells could be detected from 4 weeks onwards. This was confirmed in vitro, where reduced expansion of Apex1 KO BM cells was observed.APEX1 has two major functional activities, namely its nuclease activity, involved in BER, and its redox activity (also called Ref-1 function) important in reducing oxidized transcription factors and therefore implicated in transcriptional regulation. However, little is known regarding the nuclease and Ref-1 function(s) in primary adult hematopoietic cells. We therefore cultured BM HS(P)Cs for 1 week in the continuous presence of 2 distinct chemicals blocking the APEX1 nucleases, or 2 different chemicals inhibiting specifically the Ref-1 function. We demonstrated that both APEX1 functions are essential for hematopoiesis, even if the 2 functions appear to support the survival, expansion and maintenance of HS(P)Cs through different mechanisms. While the Ref-1 function was essential for proliferation (as both Ref-1 inhibitors cause cell cycle arrest) of all the lineages (including the Lin + cells), both inhibitors of the nuclease function affected more the expansion/survival of the less committed HS(P)Cs without leading to any cell cycle arrest.In conclusion, this study demonstrates for the first time the important role of BER genes in adult hematopoiesis, often deregulated in cancer, including hematopoietic malignancies. We observed a particularly severe phenotype upon loss of Apex1 in adult HSPCs, and ongoing studies (such as RNA sequencing analysis) should provide novel insights in underlying mechanisms of APEX1 deficiencies in HS(P)Cs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Methylmercury chronic exposure affects the expression of DNA single-strand break repair genes, induces oxidative stress, and chromosomal abnormalities in young dyslipidemic APOE knockout mice

Mercury (Hg) is one of the most toxic environmental pollutants, especially when methylated, forming methylmercury (MeHg). MeHg affects DNA repair, increases oxidative stress, and predisposes to cancer. MeHg neurotoxicity is well-known, but recently MeHg-associated cardiovascular effects were recognized. This study evaluated circulating lipids, oxidative stress, and genotoxicity after MeHg-chronic exposure (20 mg/L in drinking water) in C57BL/6J wild-type and APOE knockout (ko) mice, the latter, being spontaneously dyslipidemic. Experimental mice were assigned to four groups: non-intoxicated and MeHg-intoxicated wild-type mice and non-intoxicated and MeHg-intoxicated APOE ko mice. Plasma levels of triglycerides, total cholesterol (TC), HDL, and LDL were analyzed. Liver lipid peroxidation and splenic gene expression of xeroderma pigmentosum complementation groups A, C, D, and G (XPA, XPC, XPD, and XPG), X-ray repair cross-complementing protein 1 (XRCC1), and telomerase reverse transcriptase (TERT) were measured. Fur Hg levels confirmed chronic MeHg intoxication. MeHg exposure raises TC levels both in wild-type and APOE ko mice. HDL and LDL-cholesterol levels were increased only in the MeHg-challenged APOE ko mice. MeHg increased liver lipid peroxidation, regardless of the genetic background. Unintoxicated APOE ko mice showed higher expression of TERT than all other groups. APOE deficiency increases XPA expression, regardless of MeHg intoxication. Furthermore, MeHg-intoxicated mice had more cytogenetic abnormalities, effect which was independent of APOE deficiency. More studies are needed to dissect the interactions between circulating lipids, MeHg intoxication, and DNA-repair pathways even at young age, interactions that likely play critical roles in cell senescence and the risk for chronic disorders later in life.

Autopolyploidization affects transcript patterns and gene targeting frequencies in Physcomitrella

Key messageIn Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency.Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA–DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA–DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.

Concurrent Treatment with Pim Kinase Inhibitor Downregulates Alternative Non-Homologous End-Joining Repair and Decreases Genomic Instability in FLT3-ITD Cells Treated with Topoisomerase 2 Inhibitor Chemotherapy Drugs

Internal tandem duplication of fms -like tyrosine like kinase 3 (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML) cases, causing constitutive FLT3 signaling. While FLT3-ITD AML patients generally achieve remission following combination chemotherapy including cytarabine and a topoisomerase (topo) 2 inhibitor, they relapse rapidly, and FLT3-ITD AML cells frequently have new structural chromosome changes at relapse. Genomic instability in FLT3-ITD AML appears to result from increased generation of reactive oxygen species, which cause DNA double-strand breaks (DSBs), and upregulated DNA DSB repair by an error-prone alternative non-homologous end-joining (Alt-NHEJ) pathway.Pim-1 is an oncogenic serine/threonine kinase that is transcriptionally upregulated downstream of FLT3-ITD, contributes directly to the proliferative and anti-apoptotic effects of FLT3-ITD, and also phosphorylates and stabilizes FLT3, creating a positive feedback loop in FLT3-ITD cells. Pim kinase inhibitors are in clinical trials. We previously showed that concurrent treatment with a Pim kinase inhibitor enhances apoptosis induction by topo 2 inhibitors in FLT3-ITD cells. Here, we tested the effect of concurrent Pim kinase inhibitor treatment on Alt-NHEJ repair of topo 2 inhibitor-induced DNA DSBs and on genomic instability.Functional changes in DSB repair activities induced by treatment with the topo 2 inhibitor daunorubicin (DNR) in the absence and presence of the pan-Pim kinase inhibitor AZD1208 were studied in Ba/F3-ITD cells, with FLT3-ITD, using stably integrated green fluorescent protein (GFP)-based DSB repair reporter constructs DR-GFP, EJ5-GFP and EJ2-GFP, measuring homologous recombination (HR), classical (C-)NHEJ and Alt-NHEJ, respectively. DNR, which induced DNA DSBs, did not induce HR or C-NHEJ repair activity, but induced a 50% increase in Alt-NHEJ activity at 36 hours. Concurrent treatment with AZD1208, which increased DNR-induced DNA DSBs more than two-fold, completely abrogated the DNR-induced increase in Alt-NHEJ repair activity. Treatment of Ba/F3-ITD cells with the topo 2 inhibitors mitoxantrone (MXR) or etoposide (VP-16) also induced Alt-NHEJ repair activity, which was abrogated by concurrent treatment with AZD1208.We next studied mechanisms underlying Pim kinase inhibitor downregulation of Alt-NHEJ. The effects of DNR, MXR or VP-16 on cellular and nuclear expression of the Alt-NHEJ proteins poly (ADP-ribose) polymerase (PARP) 1, DNA polymerase θ, DNA ligase 3 and co-factor X-ray repair cross-complementing protein 1 (XRCC1) were measured in the FLT3-ITD cell lines Ba/F3-ITD, 32D-ITD, MV4-11 and MOLM-14 in the absence and presence of AZD1208. Cellular and nuclear expression of XRCC1 and DNA ligase 3, but not PARP or DNA polymerase θ, decreased in cells co-treated with AZD1208 and topo 2 inhibitors, compared to topo 2 inhibitors alone. XRCC1 was identified as a potential direct Pim-1 substrate by global profiling using the reverse in-gel kinase assay, and was confirmed as a robust Pim substrate in a reverse in-gel kinase assay. This provides a potential mechanistic link between Pim-1 kinase and ALT NHEJ.Alt-NHEJ repair is associated with genomic instability characterized by increased chromosome deletions, insertions and translocations. We studied induction of chromosome changes by topo 2 inhibitor treatment with and without Pim kinase inhibitor in FLT3-ITD cells. Ba/F3-ITD and 32D-ITD cells were incubated with DNR and/or AZD1208, or DMSO control, for 36 hours, then prepared for cytogenetic analysis.For each treatment, 20 consecutive metaphases were analyzed for induction of chromatid breaks and exchanges. No chromosome changes were seen in either cell line with DMSO or AZD1208 treatment. In Ba/F3-ITD cells, chromosome abnormalities were seen in 17 of 20 metaphases after DNR treatment, but only 8 of 20 after combination treatment (p=0.008). In 32D-ITD cells, chromosome abnormalities were seen in 8 of 20 metaphases with DNR, and 0 metaphases with combination (p=0.003).Thus concurrent treatment with Pim kinase inhibitor abrogates induction of Alt-NHEJ repair by topo 2 inhibitor treatment in FLT3-ITD cells and decreases the genomic instability that may contribute to rapid relapse of FLT3-ITD AML after chemotherapy. The data support further preclinical and clinical testing of Pim kinase inhibitors with chemotherapy in AML with FLT3-ITD. DisclosuresTron:AstraZeneca: Employment, Equity Ownership.

(5'S) 5',8-Cyclo-2'-Deoxyadenosine Cannot Stop BER. Clustered DNA Lesion Studies.

As a result of external and endocellular physical-chemical factors, every day approximately ~105 DNA lesions might be formed in each human cell. During evolution, living organisms have developed numerous repair systems, of which Base Excision Repair (BER) is the most common. 5′,8-cyclo-2′-deoxyadenosine (cdA) is a tandem lesion that is removed by the Nucleotide Excision Repair (NER) mechanism. Previously, it was assumed that BER machinery was not able to remove (5′S)cdA from the genome. In this study; however, it has been demonstrated that, if (5′S)cdA is a part of a single-stranded clustered DNA lesion, it can be removed from ds-DNA by BER. The above is theoretically possible in two cases: (A) When, during repair, clustered lesions form Okazaki-like fragments; or (B) when the (5′S)cdA moiety is located in the oligonucleotide strand on the 3′-end side of the adjacent DNA damage site, but not when it appears at the opposite 5′-end side. To explain this phenomenon, pure enzymes involved in BER were used (polymerase β (Polβ), a Proliferating Cell Nuclear Antigen (PCNA), and the X-Ray Repair Cross-Complementing Protein 1 (XRCC1)), as well as the Nuclear Extract (NE) from xrs5 cells. It has been found that Polβ can effectively elongate the primer strand in the presence of XRCC1 or PCNA. Moreover, supplementation of the NE from xrs5 cells with Polβ (artificial Polβ overexpression) forced oligonucleotide repair via BER in all the discussed cases.

Gene polymorphisms in odontogenic keratocysts and ameloblastomas: A systematic review.

The aim of this systematic review was to critically analyze available data on gene polymorphisms in odontogenic keratocysts (OKC) and ameloblastomas, including their possible relationship with clinical and histological features of these lesions. A comprehensive search of Web of Science Scopus, PubMed, Cochrane Central Register of Controlled Trials and EMBASE was conducted using relevant key terms and supplemented by a gray literature search. Quality assessment of included studies was performed using criteria from the Strengthening the Reporting of Genetic Association (STREGA) statement. Ten studies were included in the final review. Survivin -31G/C, interleukin IL-1α -889 C/T, p53 codon 72 G/C, tumor necrosis factor TNF-α (-308G>A) and its receptor TNF-R1 (36A>G), glioma-associated oncogene homolog 1 rs2228224 and matrix metalloproteinase 2 rs243865 gene polymorphisms were reported to be associated with OKC. For ameloblastomas, p53 codon 72 G/C, X-ray repair cross-complementing protein 1-codons 194 and 399 and matrix metalloproteinase 9 rs3918242 gene polymorphisms were identified as risk factors. It was not possible to establish a relationship between specific polymorphisms and clinical and histological features of investigated lesions. Several gene polymorphisms might be considered as a risk factor for the development of these lesions. Future studies should investigate whether these polymorphisms might be used to identify patients with increased risk of recurrence or aggressive disease.