Repair Of DNA Lesions Research Articles

The Cockayne syndrome protein B is involved in the repair of 5-AZA-2'-deoxycytidine-induced DNA lesions.

The Cockayne Syndrome Protein B (CSB) plays an essential role in Transcription-Coupled Nucleotide Excision Repair (TC-NER) by recruiting repair proteins once transcription is blocked with a DNA lesion. In fact, CSB-deficient cells are unable to recover from transcription-blocking DNA lesions. 5-Aza-2′-deoxycytidine (5-azadC) is a nucleoside analogue that covalently traps DNA methyltransferases (DNMTs) onto DNA. This anticancer drug has a double mechanism of action: it reverts aberrant hypermethylation in tumour-suppressor genes, and it induces DNA damage. We have recently reported that Homologous Recombination and XRCC1/PARP play an important role in the repair of 5-azadC-induced DNA damage. However, the mechanisms involved in the repair of the DNMT adducts induced by azadC remain poorly understood. In this paper, we show for the first time the importance of CSB in the repair of azadC-induced DNA lesions. We propose a model in which CSB initiates a signalling pathway to repair transcription blocks induced by incorporated 5-azadC. Indeed, CSB-deficient cells treated with 5-azadC show a delay in the repair of trapped DNMT1, increased levels of DNA damage and reduced survival.

Excited-State Decay Pathways of Flavin Molecules in Five Redox Forms: The Role of Conical Intersections.

Flavin molecules play an important role in light-driven biological activities. They have drawn significant interest for decades because of their rich photochemistry. In addition to the well-explored FADH- (anionic hydroquinone), which is supposed to be the only catalytic active state to repair DNA lesions, other four flavin molecules (i.e., FAD, FAD·-, FADH·, and FADH2) in three redox forms combined the redox cycle of flavins. Although extensive studies have been carried out for steady-state spectroscopic properties of five redox flavins in various proteins and solutions, the photochemistry and photophysical properties of those different redox states significantly complicate the corresponding theoretical studies. In present work, we employed the ab initio wave function based CASSCF method to systematically investigate the excited state decay pathways of flavins in five redox forms through two approaches. First, the comparison of the absorption and emission spectra from both theoretical calculation and experiment allows a detailed mapping of the transition properties of different redox states in flavins. Second, we identified four kinds of conical intersections (CIs) for five different redox states as the possible deactivation mechanisms responsible for internal conversion or intersystem crossing from the initially populated excited state. The theoretical calculations provide atomic details for the photochemical and photophysical properties of flavins on photoinduced processes. Our findings highlight the indispensable effects of CIs in the excited state decay of flavin molecules and thereby provide basic theoretical information for light-driven biological activities.

Influence of chromatin remodeling in the removal of UVC-induced damage in TCR proficient and deficient Chinese hamster cells

In mammalian cells, nucleotide excision repair system is constituted of two sub-pathways, global genomic repair (GGR) and transcription coupled repair (TCR). Deficiency of TCR pathway leads to Cockyane syndrome (CS) which is a rare human autosomal recessive disorder. Owing to the pivotal role of CSB gene in TCR, it’s mutation causes severe repair and transcriptional defects in CSB patients. CSB protein belongs to the ATP chromatin remodeling complex, hence presumably an improper chromatin remodeling in CSB cells could be at the source of inefficient removal of pyrimidine dimers (CPDs) after UVC exposure in these patients. In this study, we evaluated the role of chromatin remodeling process on UVC induced CPDs and the ensuing effect on chromosomal aberrations in UV61 cells (TCR deficient) and its parental cell line, AA8 (TCR proficient). We observed that post 2 h UVC irradiation, both cell lines underwent pronounced chromatin relaxation but was lower in CSB deficient UV61 cells. Since the deficiency in chromatin remodeling in CSB-mutated cells was accompanied by a decrease in the histone acetylation level, the histone deacetylase inhibitor trichostatin A (TSA) was employed to improve the removal of UVC-induced lesions by increasing the histone acetylation level. Contrary to expectations, TSA increased the induction of chromosomal aberrations and apoptotic cells along with amounts of CPDs after UVC-irradiation, indicating that changes in histone acetylation levels might contribute to the failure in the removal of UVC-induced lesions. Also, it has been shown earlier that the expression of genes regulated by CSB is affected by the increase in the acetylation level produced by TSA. Taken all together, we hypothesize that failure in the removal of UVC induced lesions in CSB-deficient cells can be caused by an imbalance in histone acetylation levels leading to chromatin conformation changes and hence interaction defects among repair proteins and DNA lesions.

Abstract A39: An integrated molecular and metabolic approach for harnessing early relapse and chemoresistance in ovarian cancer

Abstract Despite a good response rate to front-line treatment, most of the patients with epithelial ovarian cancer (EOC) will eventually develop an incurable state of platinum (Pt)-resistant disease. Pt resistance is a multifactorial event driven by different molecular mechanisms such as increased ability to tolerate or repair DNA lesions, tendency to plasticity with acquisition of mesenchymal phenotype, and ability to modify/reprogram cellular metabolism. We recently identified in EOC relevant alterations in all of these factors, largely contributing to EOC aggressiveness. Focusing our attention on microRNAs (miRNAs), the master regulator of gene expression, we identified a cluster of miRNAs located on ChrXq27.3 (miR-506 family) whose downregulation is associated with advanced-stage EOC early relapse, and we found it to be the main contributor of a 35-miRNA based predictor (MiROvaR) of EOC risk of progression/relapse. We also contributed in defining altered choline metabolism in EOC that, through overexpression of choline kinase alpha (ChoK/CHKA) enzyme whose activity sustains tumor addiction to antioxidants, fosters EOC aggressiveness also by increasing chemoresistance. By in vitro and ex vivo studies performed in human EOC cell lines, non-tumoral immortalized ovarian cells and ex vivo EOC patient-derived ascitic tumor cells (PDAT), here we analyzed the role of miR-506 in regulating molecular network associated with drug resistance and evaluated the druggability of the cholinic phenotype, exploring its possible crosstalk with the immune system. We confirmed miR-506 to directly regulate the predicted target RAD17, a gene with a key role in guiding BRCA1 to DNA damage sites and stabilizing active signaling. Since an inactivation of DNA repair genes (BRCA1/2) is associated with better prognosis due to sensitization to DNA-damaging drugs, we investigated whether miR-506 can regulate DNA repair machinery by phenocopying/supporting the effects of BRCA1/2 mutation on Pt and PARP inhibitor sensitivity. We observed that miR-506 forced expression or RAD17 knockdown sensitized EOC cells to Pt and PARP inhibitors and, interestingly, showed a synthetic lethal effect with inhibitors of cell-cycle checkpoint kinases CHK1 and WEE1. Association of miR-506 cluster expression (RT-qPCR) with the BRCAness-phenotype is currently ongoing on a retrospective FFPE case material. We also recently link EOC cells plasticity, miRNAs expression, and Pt-sensitivity. Indeed, E-cadherin can exert a new and unexpected functional role in EOC cells by contributing to Pt susceptibility. We determined that overexpression of PLEKHA7 (Pleckstrin homology domain-containing family A member 7), a component of the zonula adherens in polarized epithelia and expressed at low levels in EOC biopsies, inhibits cell tumorigenicity and sensitizes EOC cells to Pt treatment. We will evaluate PLEKHA7 ability to regulate miRNAs driving tumor progression, including some of the miR-506 family. Concerning chemoresistance associated with metabolic rewiring, we showed that CHKA silencing altered the cellular oxidative stress redox status, decreasing glutathione content and raising reactive oxygen species (ROS) levels. Interestingly, the increase in intracellular ROS species improved EOC cells’ sensitivity to Pt/taxane treatments, contributing to cell death triggering. Moreover, upon CHKA silencing both in vitro and in PDAT cells, we observed an enhanced sensitization of cancer cells to TRAIL-mediated citotoxicity due to an augmented expression of pro-apoptotic/reduced expression of anti-apoptotic TRAIL receptor members, thus further increasing tumor cells’ weakness. Exploiting tumor’s reliance on these altered molecular and metabolic pathways is expected to be an appropriate strategy to improve efficacy of current treatment, possibly circumventing drug resistance. Funded by CARIPLO Foundation, AIRC and 5x1000 Institutional Research Funding. Citation Format: Marina Bagnoli, Roberta Nicoletti, Andrea Rizzo, Francesca Roggiani, Alessandro Satta, Mariangela Figini, Wei Zhang, Panagiotis Z. Anastasiadis, Egidio Iorio, Silvana Canevari, Antonella Tomassetti, Francesco Raspagliesi, Delia Mezzanzanica. An integrated molecular and metabolic approach for harnessing early relapse and chemoresistance in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A39.

Synthesis and evaluation of aryliden- and hetarylidenfuranone derivatives of usnic acid as highly potent Tdp1 inhibitors

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a repair enzyme for stalled DNA-topoisomerase 1 (Top 1) cleavage complexes and other 3′-end DNA lesions. Tdp1 is a promising target for anticancer therapy, since it can repair DNA lesions caused by Top1 inhibitors leading to drug resistance. Hence, Tdp1 inhibition should result in synergistic effect with Top1 inhibitors. Twenty nine derivatives of (+)-usnic acid were tested for in vitro Tdp1 inhibitory activity using a fluorescent-based assay. Excellent activity was obtained, with derivative 6m demonstrating the lowest IC50 value of 25 nM. The established efficacy was verified using a gel-based assay, which gave close results to that of the fluorescent assay. In addition, molecular modeling in the Tdp1 substrate binding pocket suggested plausible binding modes for the active analogues. The synergistic effect of the Tdp1 inhibitors with topotecan, a Top1 poison in clinical use, was tested in two human cell lines, A-549 and HEK-293. Compounds 6k and 6x gave very promising results. In particular, 6x has a low cytotoxicity and an IC50 value of 63 nM, making it a valuable lead compound for the development of potent Tdp1 inhibitors for clinical use.

Neonatal Ogg1/Mutyh knockout mice have altered inflammatory gene response compared to wildtype mice in the brain and lung after hypoxia-reoxygenation.

Background 8-Oxoguanine DNA-glycosylase 1 (OGG1) and mutY DNA glycosylase (MUTYH) are crucial in the repair of the oxidative DNA lesion 7,8-dihydro-8-oxoguanine caused by hypoxia-reoxygenation injury. Our objective was to compare the gene expression changes after hypoxia-reoxygenation in neonatal Ogg1-Mutyh double knockout mice (OM) and wildtype mice (WT), and study the gene response in OM after hyperoxic reoxygenation compared to normoxic. Methods Postnatal day 7 mice were subjected to 2 h of hypoxia (8% O2) followed by reoxygenation in either 60% O2 or air, and sacrificed right after completed reoxygenation (T0h) or after 72 h (T72h). The gene expression of 44 a priori selected genes was examined in the hippocampus/striatum and lung. Results We found that OM had an altered gene response compared to WT in 21 genes in the brain and 24 genes in the lung. OM had a lower expression than WT of inflammatory genes in the brain at T0h, and higher expression at T72h in both the brain and lung. In the lung of OM, five genes were differentially expressed after hyperoxic reoxygenation compared to normoxic. Conclusion For the first time, we report that Ogg1 and Mutyh in combination protect against late inflammatory gene activation in the hippocampus/striatum and lung after neonatal hypoxia-reoxygenation.

Abstract 5041: Repair of azoxymethane-induced nuclear and mitochondrial DNA lesions in mouse liver

Abstract Background & Objectives: Azoxymethane (AOM) is a carcinogen that induces DNA lesions mainly repaired by the base excision repair (BER) pathway. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central component of BER. Mice with homozygous deletion of the Apex1 gene (which encodes APE1) cannot survive development but heterozygous animals (Apex1+/-) have a normal lifespan. Our aim was to study the role of APE1 in the repair of DNA lesions and changes in mitochondrial DNA (mtDNA) abundance induced by AOM in the liver. Methods: We isolated DNA from 6-month-old C57BL/6 WT and Apex1+/- mice that were treated with a single AOM dose (10 mg/kg) and sacrificed 24, 48 and 72 hours after treatment. To quantify nuclear DNA (nDNA) lesions, mtDNA lesions and changes in mtDNA abundance in liver tissue we applied a PCR based assay (QPCR). Results: Our data show that 24h after AOM treatment both WT and Apex1+/- mice exhibit significant levels of nDNA and mtDNA damage. The lesion number decreases 48 and 72h after treatment but remains statistically elevated as compared to their respective controls. A two tailed ANOVA analysis shows no significant differences in DNA lesions between genotypes. However, Apex1+/- mice show a statistically significant 13% decrease in mtDNA abundance 72 hours after treatment. Conclusions: These results indicate that the liver is a major target of AOM and that APE1 plays a role in preventing AOM-induced loss of mtDNA abundance. Acknowledgments: Supported by R25GM061838, 2G12RR003051 and U54CA096297. Citation Format: Carlos A. Torres-Ramos, Adlin Rodriguez-Munoz, Joan Ballista-Hernandez, Ceidy Torres-Ortiz, Sylvette Ayala-Pena. Repair of azoxymethane-induced nuclear and mitochondrial DNA lesions in mouse liver [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5041.

CFTR Deletion Confers Mitochondrial Dysfunction and Disrupts Lipid Homeostasis in Intestinal Epithelial Cells

Background: Cystic Fibrosis (CF) is a genetic disease in which the intestine exhibits oxidative and inflammatory markers. As mitochondria are the central source and the main target of reactive oxygen species, we hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) defect leads to the disruption of cellular lipid homeostasis, which contributes to mitochondrial dysfunction. Methods. Mitochondrial functions and lipid metabolism were investigated in Caco-2/15 cells with CFTR knockout (CFTR-/-) engineered by the zinc finger nuclease technique. Experiments were performed under basal conditions and after the addition of the pro-oxidant iron-ascorbate (Fe/Asc) complex. Results. Mitochondria of intestinal cells with CFTR-/-, spontaneously showed an altered redox homeostasis characterised by a significant decrease in the expression of PPARα and nuclear factor like 2. Consistent with these observations, 8-oxoguanine-DNA glycosylase, responsible for repair of ROS-induced DNA lesion, was weakly expressed in CFTR-/- cells. Moreover, disturbed fatty acid β-oxidation process was evidenced by the reduced expression of CPT1 and acyl-CoA dehydrogenase long-chain in CFTR-/- cells. The decline of mitochondrial cytochrome c and B-cell lymphoma 2 expression pointing to magnified apoptosis. Mitochondrial respiration was also affected as demonstrated by the low expression of respiratory oxidative phosphorylation (OXPHOS) complexes and a high adenosine diphosphate/adenosine triphosphate ratio. In contrast, the FAS and ACC enzymes were markedly increased, thereby indicating lipogenesis stimulation. This was associated with an augmented secretion of lipids, lipoproteins and apolipoproteins in CFTR-/- cells. The addition of Fe/Asc worsened while butylated hydroxy toluene partially improved these processes. Conclusions: CFTR silencing results in lipid homeostasis disruption and mitochondrial dysfunction in intestinal epithelial cells. Further investigation is needed to elucidate the mechanisms underlying the marked abnormalities in response to CFTR deletion.

A time-dependent fluorescent biosensor for uracil-DNA glycosylase detection based on the uracil inhibition effect towards archaebacterial DNA polymerases

Uracil-DNA glycosylase (UDG) plays important role in DNA lesion repair. It can remove the uracil lesion in DNA and is involved in multiple biological processes. UDG activity detection methods based on DNA probe are developed fast recently due to its high sensitivity and simplicity. They typically use two strategies: the conformational change or the local environmental change of the DNA probe after UDG cleavage. Here we proposed a new strategy for UDG activity detection, which utilized uracil-containing oligo to inhibit archaebacterial DNA polymerases, and after UDG treatment the archaebacterial DNA polymerases could be activated to generate linear or exponential amplification fluorescent signal. The exponential methods showed a time-dependent signal output and achieved limit of detection of 0.0025 U/mL. More importantly, the feature of time-dependence allowed us to easily modulate the resolution or the dynamic range of our method to meet different demands in various occasions. The strategy was also demonstrated to be highly selective toward DNase and exonucleases and had been applied to detect the UDG activity in cell lysate.

Carboplatin in BRCA1/2-mutated and triple-negative breast cancer BRCAness subgroups: the TNT Trial

Germline mutations in BRCA1/2 predispose individuals to breast cancer (termed germline-mutated BRCA1/2 breast cancer, gBRCA-BC) by impairing homologous recombination (HR) and causing genomic instability. HR also repairs DNA lesions caused by platinum agents and PARP inhibitors. Triple-negative breast cancers (TNBCs) harbor subpopulations with BRCA1/2 mutations, hypothesized to be especially platinum-sensitive. Cancers in putative 'BRCAness' subgroups-tumors with BRCA1 methylation; low levels of BRCA1 mRNA (BRCA1 mRNA-low); or mutational signatures for HR deficiency and those withbasal phenotypes-may also be sensitive to platinum. We assessed the efficacy of carboplatin and another mechanistically distinct therapy, docetaxel, in a phase 3 trial in subjects with unselected advanced TNBC. A prespecified protocol enabled biomarker-treatment interaction analyses in gBRCA-BC and BRCAness subgroups. The primary endpoint was objective response rate (ORR). In the unselected population (376 subjects; 188 carboplatin, 188 docetaxel), carboplatin was not more active than docetaxel (ORR, 31.4% versus 34.0%, respectively; P = 0.66). In contrast, in subjects with gBRCA-BC, carboplatin had double the ORR of docetaxel (68% versus 33%, respectively; biomarker, treatment interactionP = 0.01). Such benefit was not observed for subjects with BRCA1 methylation, BRCA1 mRNA-low tumors or a high score in a Myriad HRD assay. Significant interaction between treatment and the basal-like subtype was driven by high docetaxel response in the nonbasal subgroup. We conclude that patients with advanced TNBC benefit from characterization of BRCA1/2 mutations, but not BRCA1 methylation or MyriadHRD analyses, to inform choices on platinum-based chemotherapy. Additionally, geneexpression analysis of basal-like cancers may also influence treatment selection.

A lncRNA GUARDINg genome integrity.

A coordinated DNA damage response mediated by p53 to repair DNA lesions or to promote apoptosis is essential for maintenance of genome stability. A study now unveils the long non-coding RNA GUARDIN as a component of this pathway, which protects genome integrity in a pleiotropic fashion.

Investigation of Protein Recruitment to DNA Lesions Using 405 Nm Laser Micro-irradiation.

The DNA Damage Response (DDR) uses a plethora of proteins to detect, signal, and repair DNA lesions. Delineating this response is critical to understand genome maintenance mechanisms. Since recruitment and exchange of proteins at lesions are highly dynamic, their study requires the ability to generate DNA damage in a rapid and spatially-delimited manner. Here, we describe procedures to locally induce DNA damage in human cells using a commonly available laser-scanning confocal microscope equipped with a 405 nm laser line. Accumulation of genome maintenance factors at laser stripes can be assessed by immunofluorescence (IF) or in real-time using proteins tagged with fluorescent reporters. Using phosphorylated histone H2A.X (γ-H2A.X) and Replication Protein A (RPA) as markers, the method provides sufficient resolution to discriminate locally-recruited factors from those that spread on adjacent chromatin. We further provide ImageJ-based scripts to efficiently monitor the kinetics of protein relocalization at DNA damage sites. These refinements greatly simplify the study of the DDR dynamics.

Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology.

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress, CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits SIRT1 activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2 functions as a tumor suppressor and therefore its role in cancer formation and progression is still unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological activities in unstressed cells.

Integrated Modelling of Cell Responses after Irradiation for DNA-Targeted Effects and Non-Targeted Effects

Intercellular communication after ionizing radiation exposure, so-called non-targeted effects (NTEs), reduces cell survival. Here we describe an integrated cell-killing model considering NTEs and DNA damage along radiation particle tracks, known as DNA-targeted effects (TEs) based on repair kinetics of DNA damage. The proposed model was applied to a series of experimental data, i.e., signal concentration, DNA damage kinetics, cell survival curve and medium transfer bystander effects (MTBEs). To reproduce the experimental data, the model considers the following assumptions: (i) the linear-quadratic (LQ) function as absorbed dose to express the hit probability to emit cell-killing signals, (ii) the potentially repair of DNA lesions induced by NTEs, and (iii) lower efficiency of repair for the damage in NTEs than that in TEs. By comparing the model results with experimental data, we found that signal-induced DNA damage and lower repair efficiency in non-hit cells are responsible for NTE-related repair kinetics of DNA damage, cell survival curve with low-dose hyper-radiosensitivity (HRS) and MTBEs. From the standpoint of modelling, the integrated cell-killing model with the LQ relation and a different repair function for NTEs provide a reasonable signal-emission probability and a new estimation of low-dose HRS linked to DNA repair efficiency.

A Compass at Weak Magnetic Fields Using Thymine Dimer Repair.

How birds sense the variations in Earth’s magnetic field for navigation is poorly understood, although cryptochromes, proteins homologous to photolyases, have been proposed to participate in this magnetic sensing. Here, in electrochemical studies with an applied magnetic field, we monitor the repair of cyclobutane pyrimidine dimer lesions in duplex DNA by photolyase, mutants of photolyase, and a modified cryptochrome. We find that the yield of dimer repair is dependent on the strength and angle of the applied magnetic field even when using magnetic fields weaker than 1 gauss. This high sensitivity to weak magnetic fields depends upon a fast radical pair reaction on the thymines leading to repair. These data illustrate chemically how cyclobutane pyrimidine dimer repair may be used in a biological compass informed by variations in Earth’s magnetic field.

Oligomer formation and G-quadruplex binding by purified murine Rif1 protein, a key organizer of higher-order chromatin architecture

Rap1-interacting protein 1 (Rif1) regulates telomere length in budding yeast. We previously reported that, in metazoans and fission yeast, Rif1 also plays pivotal roles in controlling genome-wide DNA replication timing. We proposed that Rif1 may assemble chromatin compartments that contain specific replication-timing domains by promoting chromatin loop formation. Rif1 also is involved in DNA lesion repair, restart after replication fork collapse, anti-apoptosis activities, replicative senescence, and transcriptional regulation. Although multiple physiological functions of Rif1 have been characterized, biochemical and structural information on mammalian Rif1 is limited, mainly because of difficulties in purifying the full-length protein. Here, we expressed and purified the 2418-amino-acid-long, full-length murine Rif1 as well as its partially truncated variants in human 293T cells. Hydrodynamic analyses indicated that Rif1 forms elongated or extended homo-oligomers in solution, consistent with the presence of a HEAT-type helical repeat segment known to adopt an elongated shape. We also observed that the purified murine Rif1 bound G-quadruplex (G4) DNA with high specificity and affinity, as was previously shown for Rif1 from fission yeast. Both the N-terminal (HEAT-repeat) and C-terminal segments were involved in oligomer formation and specifically bound G4 DNA, and the central intrinsically disordered polypeptide segment increased the affinity for G4. Of note, pulldown assays revealed that Rif1 simultaneously binds multiple G4 molecules. Our findings support a model in which Rif1 modulates chromatin loop structures through binding to multiple G4 assemblies and by holding chromatin fibers together.

Determinants of Photolyase's DNA Repair Mechanism in Mesophiles and Extremophiles.

Light-driven DNA repair by extremophilic photolyases is of tremendous importance for understanding the early development of life on Earth. The mechanism for flavin adenine dinucleotide repair of DNA lesions is the subject of debate and has been studied mainly in mesophilic species. In particular, the role of adenine in the repair process is poorly understood. Using molecular docking, molecular dynamics simulations, electronic structure calculations, and electron tunneling pathways analysis, we examined adenine's role in DNA repair in four photolyases that thrive at different temperatures. Our results indicate that the contribution of adenine to the electronic coupling between the flavin and the cyclobutane pyrimidine dimer lesion to be repaired is significant in three (one mesophilic and two extremophilic) of the four enzymes studied. Our analysis suggests that thermophilic and hyperthermophilic photolyases have evolved structurally to preserve the functional position (and thus the catalytic function) of adenine at their high temperatures of operation. Water molecules can compete with adenine in establishing the strongest coupling pathway for the electron transfer repair process, but the adenine contribution remains substantial. The present study also reconciles prior seemingly contradictory conclusions on the role of adenine in mesophile electron transfer repair reactions, showing how adenine-mediated superexchange is conformationally gated.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end‐joining (NHEJ) pathway repairs double‐strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B‐cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA‐PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx‐knockout mice with wild‐type (WT) and NHEJ‐deficient controls including Ku80‐ and Dna‐pkcs‐null and severe combined immunodeficiency mice. Surprisingly, Paxx‐deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx‐deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx‐deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Label-Free Sensing of Human 8-Oxoguanine DNA Glycosylase Activity with a Nanopore.

Human 8-oxoguanine DNA glycosylase (hOGG1) plays a significant role in maintaining the genomic integrity of living organisms for its capability of repairing DNA lesions. Accurate detection of hOGG1 activity would greatly facilitate the screening and early diagnosis of diseases. In this work, we report a nanopore-based sensing strategy to probe the hOGG1 activity by employing the enzyme-catalytic cleavage reaction of DNA substrate. The hOGG1 specifically catalyzed the removal of the 8-hydroxyguanine (8-oxoG) and cleaved the DNA substrates immobilized on magnetic beads, thereby releasing the output DNA which would quantitatively produce the signature current events when subjected to α-hemolysin (α-HL) nanopore test. The approach enables the sensitive detection of hOGG1 activity without the need of any labeling or signal amplification route. Furthermore, the method can be applied to assay the inhibition of hOGG1 and evaluate the activity of endogenous hOGG1 in crude cell extracts. Importantly, since DNAs with specific sequences are the catalytic substrates of a wide variety of enzymes, the proposed strategy should be universally applicable for probing the activities of different types of enzymes with nanopore sensors.

Characterizing the Enhanced Nanoscale Translocation Properties of hUNG2 Facilitated by its Disordered N-terminal Domain In Vitro and in Human Cells

Nuclear localized human uracil DNA glycosylase 2 (hUNG2) initiates base excision repair (BER) of uracil lesions in genomic DNA. Similar to many DNA glycosylases, hUNG2 possesses a ∼90 amino acid unstructured N-terminal domain (NTD) that encodes the nuclear localization signal as well as several sites for post translational modification and interaction with replication proteins. Here, we present evidence that within crowded environments such as the cell nucleus, the hUNG2 NTD endows enhanced nanoscale translocation properties to the enzyme. Based on in vitro measurements comparing hUNG2 to a catalytic hUNG2 construct lacking the NTD under crowded conditions, these enhancements are manifested in a near 10-fold DNA length dependent increase in kcat, a ∼3-fold increase in the overall catalytic efficiency (kcat/KM), and an improved ability for the enzyme to translocate between damaged sites separated greater than 20 bp. These translocation studies were further implemented in human cells to develop the first in vivo assay of its kind, able to detect nanoscale translocation events of hUNG2 between uracil lesion sites 10 to 80 bp apart. hUNG2 translocation between target sites in human cells increased over that seen under similar conditions in vitro. These findings together reveal a novel role for both the hUNG2 NTD as well as the crowded nuclear environment in facilitating DNA damage detection.