DNA Base Excision Repair Pathway Research Articles

Modifying the Basicity of the dNTP Leaving Group Modulates Precatalytic Conformational Changes of DNA Polymerase β.

The catalytic function of DNA polymerase β (pol β) fulfills the gap-filling requirement of the base excision DNA repair pathway by incorporating a single nucleotide into a gapped DNA substrate resulting from the removal of damaged DNA bases. Most importantly, pol β can select the correct nucleotide from a pool of similarly structured nucleotides to incorporate into DNA in order to prevent the accumulation of mutations in the genome. Pol β is likely to employ various mechanisms for substrate selection. Here, we use dCTP analogues that have been modified at the β,γ-bridging group of the triphosphate moiety to monitor the effect of leaving group basicity of the incoming nucleotide on precatalytic conformational changes, which are important for catalysis and selectivity. It has been previously shown that there is a linear free energy relationship between leaving group pKa and the chemical transition state. Our results indicate that there is a similar relationship with the rate of a precatalytic conformational change, specifically, the closing of the fingers subdomain of pol β. In addition, by utilizing analogue β,γ-CHX stereoisomers, we identified that the orientation of the β,γ-bridging group relative to R183 is important for the rate of fingers closing, which directly influences chemistry.

Abstract 4527: TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress

Abstract Defective maintenance of genomic integrity is a hallmark of cancer cells that can result from oncogene-induced replication stress and by loss of DNA repair mechanisms. DNA repair deficiencies and elevated replication stress present targetable vulnerabilities for cancer treatment. Notably, BRCA1/2 mutant and homologous recombination deficient (HRD) tumors cannot repair double-strand breaks by homologous recombination and rely on alternative pathways of DNA repair. PARP inhibitors (PARPi), which are a standard of care in many BRCA1/2 mutant tumors, cause synthetic lethality with BRCA1/2 mutation by inhibiting the DNA base excision repair pathway. Despite the clinical benefit of PARPi, they are not effective in every HRD tumor and the acquisition of PARPi resistance limits long-term response. TNG348, a selective allosteric inhibitor of the deubiquitinating enzyme USP1, was specifically designed to target HRD vulnerabilities through an alternative mechanism. We previously showed that the anti-tumor activity of USP1 inhibition results from disruption of the translesion synthesis DNA damage tolerance pathway, a mechanism of action that is functionally distinct from base excision repair targeted by PARPi. Our preclinical studies show that TNG348 is active in HRD models and strongly synergizes with PARP inhibitors to drive strong anti-tumor responses. We have identified replication stress as a predictive biomarker of TNG348 response using cell line profiling and genome-wide CRISPR screens. For example, overexpression of oncogenes known to induce replication stress sensitized to the TNG348 and PARPi combination both in vitro and in vivo. These data indicate that cancer-specific elevated DNA replication stress could contribute to tumor sensitivity to TNG348 and provide additional patient stratification strategies and opportunities for indication expansion. Citation Format: Antoine Simoneau, Hsin-Jung Wu, Charlotte Pratt, Grace Comer, Shangtao Liu, Samuel Meier, Tenzing Khendu, Ashley Choi, Hongxiang Zhang, Binzhang Shen, Douglas Whittington, Sirimas Sudsakorn, Wenhai Zhang, Yi Yu, Yingnan Chen, Brian Haines, Adam Crystal, Jannik N. Andersen, John Maxwell, Scott Throner. TNG348 is synergistic with PARP inhibitors in tumor models with elevated replication stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4527.

DNA polymerase beta connects tumorigenicity with the circadian clock in liver cancer through the epigenetic demethylation of Per1

The circadian-controlled DNA repair exhibits a strong diurnal rhythm. Disruption in circadian clock and DNA repair is closely linked with hepatocellular carcinoma (HCC) progression, but the mechanism remains unknown. Here, we show that polymerase beta (POLB), a critical enzyme in the DNA base excision repair pathway, is rhythmically expressed at the translational level in mouse livers. Hepatic POLB dysfunction dampens clock homeostasis, whereas retards HCC progression, by mediating the methylation of the 4th CpG island on the 5′UTR of clock gene Per1. Clinically, POLB is overexpressed in human HCC samples and positively associated with poor prognosis. Furthermore, the hepatic rhythmicity of POLB protein expression is orchestrated by Calreticulin (CALR). Our findings provide important insights into the molecular mechanism underlying the synergy between clock and food signals on the POLB-driven BER system and reveal new clock-dependent carcinogenetic effects of POLB. Therefore, chronobiological modulation of POLB may help to promote precise interventions for HCC.

OGG1 as an Epigenetic Reader Affects NFκB: What This Means for Cancer.

8-oxoguanine glycosylase 1 (OGG1), which was initially identified as the enzyme that catalyzes the first step in the DNA base excision repair pathway, is now also recognized as a modulator of gene expression. What is important for cancer is that OGG1 acts as a modulator of NFκB-driven gene expression. Specifically, oxidant stress in the cell transiently halts enzymatic activity of substrate-bound OGG1. The stalled OGG1 facilitates DNA binding of transactivators, such as NFκB to their cognate sites, enabling the expression of cytokines and chemokines, with ensuing recruitment of inflammatory cells. Recently, we highlighted chief aspects of OGG1 involvement in regulation of gene expression, which hold significance in lung cancer development. However, OGG1 has also been implicated in the molecular underpinning of acute myeloid leukemia. This review analyzes and discusses how these cells adapt through redox-modulated intricate connections, via interaction of OGG1 with NFκB, which provides malignant cells with alternative molecular pathways to transform their microenvironment, enabling adjustment, promoting cell proliferation, metastasis, and evading killing by therapeutic agents.

Structural basis of nuclear transport for NEIL DNA glycosylases mediated by importin-alpha

NEIL glycosylases, including NEIL1, NEIL2, and NEIL3, play a crucial role in the base excision DNA repair pathway (BER). The classical importin pathway mediated by importin α/β and cargo proteins containing nuclear localization sequences (NLS) is the most common transport mechanism of DNA repair proteins to the nucleus. Previous studies have identified putative NLSs located at the C-terminus of NEIL3 and NEIL1. Crystallographic, bioinformatics, calorimetric (ITC), and fluorescence assays were used to investigate the interaction between NEIL1 and NEIL3 putative NLSs and importin-α (Impα). Our findings showed that NEIL3 contains a typical cNLS, with medium affinity for the major binding site of Impα. In contrast, crystallographic analysis of NEIL1 NLS revealed its binding to Impα, but with high B-factors and a lack of electron density at the linker region. ITC and fluorescence assays indicated no detectable affinity between NEIL1 NLS and Impα. These data suggest that NEIL1 NLS is a non-classical NLS with low affinity to Impα. Additionally, we compared the binding mode of NEIL3 and NEIL1 with Mus musculus Impα to human isoforms HsImpα1 and HsImpα3, which revealed interesting binding differences for HsImpα3 variant. NEIL3 is a classical medium affinity monopartite NLS, while NEIL1 is likely to be an unclassical low-affinity bipartite NLS. The base excision repair pathway is one of the primary systems involved in repairing DNA. Thus, understanding the mechanisms of nuclear transport of NEIL proteins is crucial for comprehending the role of these proteins in DNA repair and disease development.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.

Biochemical Reconstitution of the Mimiviral Base Excision Repair Pathway

Viruses are believed to be the obligate intracellular parasites that only carry genes essential for infecting and hijacking the host cell machinery. However, a recently discovered group of viruses belonging to the phylum nucleocytovirocota, also known as the nucleo-cytoplasmic large DNA viruses (NCLDVs), possess a number of genes that code for proteins predicted to be involved in metabolism, and DNA replication, and repair. In the present study, first, using proteomics of viral particles, we show that several proteins required for the completion of the DNA base excision repair (BER) pathway are packaged within the virions of Mimivirus as well as related viruses while they are absent from the virions of Marseillevirus and Kurlavirus that are NCLDVs with smaller genomes. We have thoroughly characterized three putative base excision repair enzymes from Mimivirus, a prototype NCLDV and successfully reconstituted the BER pathway using the purified recombinant proteins. The mimiviral uracil-DNA glycosylase (mvUDG) excises uracil from both ssDNA and dsDNA, a novel finding contrary to earlier studies. The putative AP-endonuclease (mvAPE) specifically cleaves at the abasic site created by the glycosylase while also exhibiting the 3′-5′ exonuclease activity. The Mimivirus polymerase X protein (mvPolX) can bind to gapped DNA substrates and perform single nucleotide gap-filling followed by downstream strand displacement. Furthermore, we show that when reconstituted in vitro, mvUDG, mvAPE, and mvPolX function cohesively to repair a uracil-containing DNA predominantly by long patch BER and together, may participate in the BER pathway during the early phase of Mimivirus life-cycle.

DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

PARP1 and POLD2 as prognostic biomarkers for multiple myeloma in autologous stem cell transplant.

Multiple Myeloma (MM) is an incurable plasma cell malignancy often treated by autologous stem cell transplant (ASCT). Clinical response to ASCT has been associated with DNA repair efficiency. Here we interrogated the role of the base excision DNA repair (BER) pathway in MM response to ASCT. Across 450 clinical samples and six disease stages, expression levels of genes in the BER pathway were found to be highly upregulated during the development of MM. In a separate cohort of 559 patients with MM treated with ASCT, expression of BER pathway members MPG and PARP3 was positively associated with overall survival (OS) while expression of PARP1, POLD1, and POLD2 was negatively associated with OS. In a validation cohort of 356 patients with MM treated with ASCT, PARP1 and POLD2 findings were replicated. In patients with MM who never received ASCT (n=319), PARP1 and POLD2 were not associated with OS, suggesting that the prognostic effect of these genes may be treatment-dependent. In preclinical models of MM, synergy was observed in anti-tumor activity when poly (ADPribose) polymerase (PARP) inhibitors (olaparib, talazoparib) were used in combination with melphalan. The negative prognosis associated with PARP1 and POLD2 expression along with the apparent melphalan-sensitizing effect of PARP inhibition may suggest this pathway as a potential biomarker in patients with MM in the setting of ASCT. Further understanding of the role of the BER pathway in MM is vital to improve therapeutic strategies related to ASCT.

CircCIMT Silencing Promotes Cadmium-Induced Malignant Transformation of Lung Epithelial Cells Through the DNA Base Excision Repair Pathway.

Changes in gene expression in lung epithelial cells are detected in cancer tissues during exposure to pollutants, highlighting the importance of gene-environmental interactions in disease. Here, a Cd-induced malignant transformation model in mouse lungs and bronchial epithelial cell lines is constructed, and differences in the expression of non-coding circRNAs are analyzed. The migratory and invasive abilities of Cd-transformed cells are suppressed by circCIMT. A significant DNA damage response is observed after exposure to Cd, which increased further following circCIMT-interference. It is found that APEX1 is significantly down-regulated following Cd exposure. Furthermore, it is demonstrated that circCIMT bound to APEX1 during Cd exposure to mediate the DNA base excision repair (BER) pathway, thereby reducing DNA damage. In addition, simultaneous knockdown of both circCIMT and APEX1 promotes the expression of cancer-related genes and malignant transformation after long-term Cd exposure. Overall, these findings emphasis the importance of genetic-epigenetic interactions in chemical-induced cancer transformation.

DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

Abstract 1283: Coupling of the two enzymatic activities of DNA polymerase beta redefine the DNA base excision repair pathway

Abstract 1283: Coupling of the two enzymatic activities of DNA polymerase beta redefine the DNA base excision repair pathway

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a β-clamp-XthA-LigA tri-component complex

The Class-II AP-endonuclease (XthA) is a mycobacterial DNA base excision repair (BER) pathway enzyme that functions in the initial steps. It acts on DNA substrates that contain abasic sites to create nicks with 3′-hydroxyl (OH) and 5′-deoxyribose phosphate (5′-dRP) moieties. The NAD+-dependent DNA ligase (LigA) is the terminal player in mycobacterial BER and seals such nicks efficiently. Here, we demonstrate that the Mtbβ-clamp-MtbXthA complex that exists in the initial steps of BER engages with MtbLigA to form a novel tri-component BER complex. Size exclusion chromatography (SEC) experiments analysis show that the three proteins interact with equimolar stoichiometry. Small angle X-ray scattering (SAXS) analysis and associated studies reveal that the apo tri-component BER-complex adopts an extended conformation where MtbXthA is sandwiched between the Mtbβ-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbβ-clamp through 239QLRFPKK245 motif and to MtbLigA by 104DGQPSWSGKP113 motif simultaneously. However, the complex adopts a less-extended conformation in the presence of substrate DNA, where MtbXthA interactions switch from predominantly subsite-I to subsite-II of the Mtbβ-clamp. Overall, the novel tri-component complex prevents futile ligation activity of MtbLigA on the product of MtbXthA and ensures forward progression of the pathway and productive mycobacterial BER interactions.

Evaluation of natural chronic low dose radiation exposure on telomere length and transcriptional response of shelterin complex in individuals residing in Kerala coast, India

The high level natural radiation areas (HLNRA) of Kerala coast provide unique opportunity to study the biological effect of chronic low dose ionizing radiation (LDIR) on human population below 100 mSv. The radiation level in this area varies from < 1.0–45 mGy /year due to patchy distribution of monazite in the sand, which contains 232Th (8–10%), 238U (0.3%), and their decay products. Telomere length attrition has been correlated to DNA damage due to genotoxic agents. The objective of the present study is to evaluate the effect of natural chronic LDIR exposure on telomere length and transcriptional response of telomere specific and DNA damage repair genes in peripheral blood mononuclear cells (PBMCs) of individuals from normal level natural radiation areas (NLNRA) and HLNRA of Kerala coast, southwest India. Blood samples were collected from 71 random male donors (24–80 years) from NLNRA (≤1.50 mGy/year; N = 19) and two HLNRA dose groups [1.51–10 mGy/year (N = 17); > 10 mGy/year, (N = 35)]. Genomic DNA was isolated from PBMCs and relative telomere length (RTL) was determined using real time q-PCR. Radio-adaptive response (RAR) study was carried out in PBMCs of 40 random males from NLNRA (N = 20) and HLNRA (>10 mGy/year; N = 20), where PBMCs were given a challenged dose of 2.0 Gy gamma radiation at 4 h. Transcriptional profile of telomere specific (TRF1, TRF2, POT1, TIN2, TPP1, RAP1), DNA damage response (RAD17, ATM, CHEK1) and base excision repair pathway (BER) (OGG1, XRCC1, NTH1, NEIL1, MUTYH, MBD4) genes were analysed at basal level and after a challenge dose of 2.0 Gy at 4 h. Our results did not show any significant effect of chronic LDR on RTL among the individuals from NLNRA and two HLNRA groups (p = 0.195). However, influence of age on RTL was clearly evident among NLNRA and HLNRA individuals. At basal level, TRF1, TRF2, TIN2, MBD4, NEIL1 and RAD17 showed significant up-regulation, whereas XRCC1 was significantly down regulated in HLNRA individuals. After a challenge dose of 2.0 Gy, significant transcriptional up-regulation was observed at telomere specific (TRF2, POT1) and BER (MBD4, NEIL1) genes in HLNRA individuals as compared to NLNRA suggesting their role in RAR. In conclusion, elevated level of natural chronic LDR exposure did not have any adverse effect on telomere length in Kerala coast. Significant transcriptional response at TRF2, MBD4 and NEIL1 at basal level and with a challenge dose of 2.0 Gy suggested their active involvement in efficient repair and telomere maintenance in individuals from HLNRA of Kerala coast.

Altered oxidative DNA damage repair systems in muscles of old mice: role in the age‐related increase in muscle production of cytokines/chemokines

An increased presence of the oxidised DNA base lesion 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) is seen in muscles of old mice. 8‐oxoG is repaired by the 8‐oxoguanine DNA glycosylase‐1 (OGG1)‐initiated DNA base excision repair pathway, a process proposed to lead to activation of nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐kB). We hypothesised that chronic oxidative DNA damage in muscles of old mice leads to the chronic increase in activation of NF‐kB by OGG1 and the subsequent production of inflammatory cytokines by skeletal muscle, contributing to age‐related deterioration of muscle function.An in vitro model of oxidative DNA damage was established by treating C2C12 myoblasts with H2O2. DNA damage repair responses including 8‐oxoG, OGG1, PARP‐1, NF‐kB activation and cytokine/chemokine gene expression and release by muscle cells were determined using Luminex multiplex analysis. The effect of TH5487 (5µM), a selective OGG1 inhibitor on NF‐kB activation and cytokine/chemokine production was determined. The role of OGG1 in the chronic production of cytokines/chemokines by isolated muscle fibres of adult mice treated with H2O2 and of old mice was examined. Cell viability was maintained in myoblasts treated with 50µM H2O2 but Ogg1 mRNA levels were significantly increased as was acetylation of OGG1, PARP‐1 and cleaved PARP‐1 protein levels. Activation of NF‐κB was evident and accompanied by an increased expression and release of a number of cytokines/chemokines (3.7 fold, 4.2 fold, 3 fold increases in IL‐6 TNF‐a and CXCL1 mRNA respectively). The H2O2‐mediated increase in NF‐kB activation and increase in cytokine/chemokine mRNA levels were normalized when cells were pre‐treated with TH5487 prior to treatment with H2O2. Experiments using isolated muscle fibers from adult mice demonstrated an increase in NF‐κB activation following treatment with H2O2. This H2O2‐mediated increase in NF‐kB DNA binding activity was significantly decreased in muscle fibers pre‐treated with TH5487. Isolated muscle fibers from old compared with adult mice showed an increase in cytokine/chemokine release which was also reduced by treatment with TH5487. Increases in IL‐6, CCL2,CXCL1,CCL7,CCL27,CXCL5,CCL11,CXCL16 and CXCL‐12 were seen in the media of fibers from adult mice following treatment with H2O2 and most of these were then decreased to normal values by pre‐treatment with TH5487. Isolated muscle fibers from old mice showed a significant increase in IL‐6, CXCL1,CCL2,CCL7, of which all were reduced following treatment with TH5487, suggesting that activation of the OGG1 pathway may be responsible, at least in part for the increased production of cytokines/chemokines by muscle of old mice. Data on proteomic and mRNA analyses of muscles from adult and old mice for evidence of changes in DNA repair pathways with age will also be presented. A computationalmodel of DNA damage repair is being designed to identify DNA damage repair process therapeutic targets to modify activation of NF‐κB in muscles of old mice.

Neuron-targeted Knockout of APE1 Forces Premature Cognitive Impairment and Synaptic Dysfunction in Adult Mice.

Adaptable and consistent neural function relies at least in part on the ongoing repair of oxidative damage that can accumulate in the brain over a lifespan. To determine whether forebrain neuron-targeted knockout of AP endonuclease 1 (APE1), a critical enzyme in the base excision DNA repair pathway, contributes to neuronal impairments, we generated APE1 conditional knockout mice under the control of the CamKIIα promotor (APE1 cKO). Spatial learning and memory were tested using the Morris water maze. Synaptic markers, including synapsin, vGLUT, GABA1, and GAD were immunostained and quantified. Dendritic morphology and number were characterized using Golgi staining. Long-term potentiation (LTP) was measured in slices from the 6-month-old brain. APE1 cKO mice did not significantly differ from WT mice in the learning phase of the Morris water maze, but performed significantly worse during the memory phase of the Morris water maze. vGLUT, GABA1, and GAD immunostaining was significantly decreased in APE1 cKO mice without concomitant changes in the number of synapsin-positive structures, suggesting that neural networks may be impaired but not at the level of total presynaptic structures. Dendrites were reduced both in number and length of spines in APE1 cKO mice. APE1 cKO brain slices exhibited decreased LTP induction compared to WT brain slices. Together, these data indicate that the conditional loss of APE1 in forebrain neurons leads to a phenotype consistent with expedited brain aging.

Mild phenotype of knockouts of the major apurinic/apyrimidinic endonuclease APEX1 in a non-cancer human cell line.

The major human apurinic/apyrimidinic (AP) site endonuclease, APEX1, is a central player in the base excision DNA repair (BER) pathway and has a role in the regulation of DNA binding by transcription factors. In vertebrates, APEX1 knockouts are embryonic lethal, and only a handful of knockout cell lines are known. To facilitate studies of multiple functions of this protein in human cells, we have used the CRISPR/Cas9 system to knock out the APEX1 gene in a widely used non-cancer hypotriploid HEK 293FT cell line. Two stable knockout lines were obtained, one carrying two single-base deletion alleles and one single-base insertion allele in exon 3, another homozygous in the single-base insertion allele. Both mutations cause a frameshift that leads to premature translation termination before the start of the protein’s catalytic domain. Both cell lines totally lacked the APEX1 protein and AP site-cleaving activity, and showed significantly lower levels of the APEX1 transcript. The APEX1-null cells were unable to support BER on uracil- or AP site-containing substrates. Phenotypically, they showed a moderately increased sensitivity to methyl methanesulfonate (MMS; ~2-fold lower EC50 compared with wild-type cells), and their background level of natural AP sites detected by the aldehyde-reactive probe was elevated ~1.5–2-fold. However, the knockout lines retained a nearly wild-type sensitivity to oxidizing agents hydrogen peroxide and potassium bromate. Interestingly, despite the increased MMS cytotoxicity, we observed no additional increase in AP sites in knockout cells upon MMS treatment, which could indicate their conversion into more toxic products in the absence of repair. Overall, the relatively mild cell phenotype in the absence of APEX1-dependent BER suggests that mammalian cells possess mechanisms of tolerance or alternative repair of AP sites. The knockout derivatives of the extensively characterized HEK 293FT cell line may provide a valuable tool for studies of APEX1 in DNA repair and beyond.

The dynamic changes of Nrf2 mediated oxidative stress, DNA damage and base excision repair in testis of rats during aging.

Accumulation of oxidative stress, DNA damage and impaired DNA repair appear to play critical roles in the decline of testicular function with aging. However, when those factors begin to lose control in testis during aging has not yet been well understood. This study was designed to assess the changes of oxidative stress and DNA damage status, and DNA repair capacity in testis during aging. Thus, male Sprague-Dawley rats at 3, 9, 15 and 24months of age were used to delineate the dynamic changes in testicular weight and index, testosterone concentration, testicular histology, Nrf2-mediated oxidative stress, DNA damage, DNA repair and apoptosis. Results showed that testicular weight and index, testosterone concentration and spermatid number progressively declined from 9 to 24months of age. Similarly, seminiferous tubule diameters and seminiferous epithelium heights gradually diminished with aging. Nrf2-mediated antioxidant defense ability was significantly impaired in testis with increasing age including decreased the activity of SOD and the expression levels of Nrf2, HO-1 and NQO-1, and increased the contents of MDA. In addition, DNA damage including DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) also progressively increased accompanied by increased levels of 8-hydroxydeoxyguanosine (8-OHdG) and γ-H2AX, and activated ATM/Chk2 and ATR/Chk1 pathway. Consistent with the results of Nrf2 pathway, the expression levels of APE1, OGG1 and XRCC1 involved in base excision DNA repair (BER) pathway increased from 3 to 9months of age, and then gradually decreased after 9months of age. Finally, TUNEL and Western blot results further confirmed germ cell apoptosis progressively increased from 3 to 24months of age as evidenced by decreased ratio of Bcl-2/Bax and levels of Bcl-2 expression, and increased Bax expression levels. Taken together, our results suggest that downregulation of antioxidant ability mediated by Nrf2 pathway and impairment of BER capacity might correlate with increased DNA damage, and then induce declining testicular function during aging after adult.

The Genomic Landscape of Radioactive Iodine Refractory and Avid Papillary Thyroid Carcinomas

Background: Although, standard treatment of Papillary Thyroid Carcinoma (PTC), involving surgery followed by radioactive iodine (RAI) therapy, is curative for most patients, 5-20% of patients develop RAI refractory disease. The repertoire of genomic events enabling RAI refractory disease in PTC needs elucidating. Methods: Whole-exome sequencing was performed on 100 primary PTC tumours, consisting of 47 RAI refractory and 53 RAI avid tumours, with matched germline. The resulting somatic variants were analysed to compare the genomic landscape, driver events and clinically actionable events between RAI refractory and avid PTC. Results: RAI refractory primary tumours were significantly associated with later stage, positive involvement of surgical margins, presence of extrathyroidal extension, lymph node metastases and poorer 5-year disease-free survival. Mutational burden was significantly higher, with additional subclonal mutations in RAI refractory compared to avid PTC patients. RAI refractory primary tumours showed significantly stronger PD-L1 expression. Mutations and PD-L1 expression in RAI Refractory PTC tumours significantly correlated with mutational signatures related to defective DNA base and nucleotide excision repair pathways. Driver mutations were acquired earlier in RAI refractory than in avid primary tumours. Conclusions: We conclude that RAI refractoriness is gained early in PTC, and is significantly associated with a higher mutational burden, PD-L1 expression, and mutational signatures, which may serve as important prognostic factors and indicate suitability for treatment with immune checkpoint inhibitors.