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Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5′ side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2′-deoxyuridine (DHU), 2′-deoxyuridine (dU), alpha-2′-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron–electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1. [ABSTRACT FROM AUTHOR]

Hyperthermophilic archaea such as Pyrococcus furiosus survive under very aggressive environmental conditions by occupying niches inaccessible to representatives of other domains of life. The ability to survive such severe living conditions must be ensured by extraordinarily efficient mechanisms of DNA processing, including repair. Therefore, in this study, we compared kinetics of conformational changes of DNA Endonuclease Q from P. furiosus during its interaction with various DNA substrates containing an analog of an apurinic/apyrimidinic site (F-site), hypoxanthine, uracil, 5,6-dihydrouracil, the α-anomer of adenosine, or 1,N6-ethenoadenosine. Our examination of DNA cleavage activity and fluorescence time courses characterizing conformational changes of the dye-labeled DNA substrates during the interaction with EndoQ revealed that the enzyme induces multiple conformational changes of DNA in the course of binding. Moreover, the obtained data suggested that the formation of the enzyme–substrate complex can proceed through dissimilar kinetic pathways, resulting in different types of DNA conformational changes, which probably allow the enzyme to perform its biological function at an extreme temperature. [ABSTRACT FROM AUTHOR]

Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polβ in BER through studying an impact of APE1 on Polβ-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polβ′s catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3′ blocking groups and corrected Polβ-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polβ not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polβ during the BER process. • APE1 affects the different steps of Polβ nucleotide incorporation into DNA. • APE1's impact on Polβ′s catalysis depends on nature of DNA substrate. • Effect of APE1 on Polβ realized by formation of ternary APE1-Polβ-DNA complex. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic endonuclease 1 (APE1) is one of the most important enzymes in base excision repair. Studies on this enzyme have been conducted for a long time, but some aspects of its activity remain poorly understood. One such question concerns the mechanism of damaged-nucleotide recognition by the enzyme, and the answer could shed light on substrate specificity control in all enzymes of this class. In the present study, by pulsed electron–electron double resonance (DEER, also known as PELDOR) spectroscopy and pre–steady-state kinetic analysis along with wild-type (WT) APE1 from Danio rerio (zAPE1) or three mutants (carrying substitution N253G, A254G, or E260A), we aimed to elucidate the molecular events in the process of damage recognition. The data revealed that the zAPE1 mutant E260A has much higher activity toward DNA substrates containing 5,6-dihydro-2′-deoxyuridine (DHU), 2′-deoxyuridine (dU), alpha-2′-deoxyadenosine (αA), or 1,N6-ethenoadenosine (εA). Examination of conformational changes in DNA clearly revealed multistep DNA rearrangements during the formation of the catalytic complex. These structural rearrangements of DNA are directly associated with the capacity of damaged DNA for enzyme-induced bending and unwinding, which are required for eversion of the damaged nucleotide from the DNA duplex and for its placement into the active site of the enzyme. Taken together, the results experimentally prove the factors that control substrate specificity of the AP endonuclease zAPE1. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) is one of the important systems for the maintenance of genome stability via repair of DNA lesions. BER is a multistep process involving a number of enzymes, including damage-specific DNA glycosylases, apurinic/apyrimidinic (AP) endonuclease 1, DNA polymerase β, and DNA ligase. Coordination of BER is implemented by multiple protein–protein interactions between BER participants. Nonetheless, mechanisms of these interactions and their roles in the BER coordination are poorly understood. Here, we report a study on Polβ's nucleotidyl transferase activity toward different DNA substrates (that mimic DNA intermediates arising during BER) in the presence of various DNA glycosylases (AAG, OGG1, NTHL1, MBD4, UNG, or SMUG1) using rapid-quench-flow and stopped-flow fluorescence approaches. It was shown that Polβ efficiently adds a single nucleotide into different types of single-strand breaks either with or without a 5′-dRP–mimicking group. The obtained data indicate that DNA glycosylases AAG, OGG1, NTHL1, MBD4, UNG, and SMUG1, but not NEIL1, enhance Polβ's activity toward the model DNA intermediates. [ABSTRACT FROM AUTHOR]

In this work, we developed a label-free and one-tube method for apurinic/apyrimidinic endonuclease 1 (APE1) sensing based on ligation initiated self-priming isothermal polymerization (SPIP) and signal amplification of fluorescent copper nanoparticles (CuNPs). This method was rationally proposed with modular design. In target recognition module, APE1 could specifically cleave substrate to release a linker DNA that served the ligation of an adenine/thymine (AT)-rich hairpin and a phosphorothioate-modified hairpin with the assistance of ligase. Then in SPIP amplification and signal transduction modules, once driven by polymerase, enzymatic extension and self-folding alternately occurred on the primary DNA originated from the ligation reaction, which resulted in accumulation of numerous AT-rich templates repeatedly included in the elongated dsDNA products, and ultimately enabled rapid formation of fluorescent CuNPs to output amplified signal. The accurate target identification and efficient signal amplification facilitated the highly selective and sensitive detection of APE1 with limit of detection of 8.6 × 10−4 U/mL. Benefiting from the robustness of this method, the normal cells and tumor cells could be distinguished unambiguously with this method according to the test results of cellular APE1. Moreover, the sensing strategy was employed in the concatenated AND logical operation and molecular keypad unlocking, which further displayed its extensibility and versatility. [Display omitted] • Self-priming isothermal polymerization enabled nano-signal amplification was realized. • A label-free, highly-sensitive and one-tube method for APE1 sensing was put forward. • Normal cells and tumor cells were distinguished unambiguously with this method. • A molecular keypad lock model was successfully built based on this sensing strategy. [ABSTRACT FROM AUTHOR]

Escherichia coli apurinic/apyrimidinic (AP) endonuclease Nfo is one of the key participants in DNA repair. The principal biological role of this enzyme is the recognition and hydrolysis of AP sites, which arise in DNA either as a result of the spontaneous hydrolysis of an N-glycosidic bond with intact nitrogenous bases or under the action of DNA glycosylases, which eliminate various damaged bases during base excision repair. Nfo also removes 3′-terminal blocking groups resulting from AP lyase activity of DNA glycosylases. Additionally, Nfo can hydrolyze the phosphodiester linkage on the 5′ side of some damaged nucleotides on the nucleotide incision repair pathway. The function of 3′-5′-exonuclease activity of Nfo remains unclear and probably consists of participation (together with the nucleotide incision repair activity) in the repair of cluster lesions. In this work, using polyacrylamide gel electrophoresis and the stopped-flow method, we analyzed the kinetics of the interaction of Nfo with various model DNA substrates containing a 5′ single-stranded region. These data helped to describe the mechanism of nucleotide cleavage and to determine the rates of the corresponding stages. It was revealed that the rate-limiting stage of the enzymatic process is a dissociation of the reaction product from the enzyme active site. The stability of the terminal pair of nucleotides in the substrate did not affect the enzymatic-reaction rate. Finally, it was found that 2′-deoxynucleoside monophosphates can effectively inhibit the 3′-5′-exonuclease activity of Nfo. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) endonucleases are the key DNA repair enzymes in the base excision repair (BER) pathway, and are responsible for hydrolyzing phosphodiester bonds on the 5′ side of an AP site. The enzymes can recognize not only AP sites but also some types of damaged bases, such as 1,N6-ethenoadenosine, α-adenosine, and 5,6-dihydrouridine. Here, to elucidate the mechanism underlying such a broad substrate specificity as that of AP endonucleases, we performed a computational study of four homologous APE1-like endonucleases: insect (Drosophila melanogaster) Rrp1, amphibian (Xenopus laevis) APE1 (xAPE1), fish (Danio rerio) APE1 (zAPE1), and human APE1 (hAPE1). The contact between the amino acid residues of the active site of each homologous APE1-like enzyme and the set of damaged DNA substrates was analyzed. A comparison of molecular dynamic simulation data with the known catalytic efficiency of these enzymes allowed us to gain a deep insight into the differences in the efficiency of the cleavage of various damaged nucleotides. The obtained data support that the amino acid residues within the "damage recognition" loop containing residues Asn222–Ala230 significantly affect the catalytic-complex formation. Moreover, every damaged nucleotide has its unique position and a specific set of interactions with the amino acid residues of the active site. [ABSTRACT FROM AUTHOR]

The DNA repair capacity plays a critical role in maintaining the genomic stability and gatekeeping for individual cancer risk. In this study, we aim at evaluation the role of the Asp148Glu (rs1130409) variant at apurinic/apyrimidinic endonuclease (APE) gene in renal cell carcinoma (RCC) risk and the contribution of different genotypes to its transcriptional mRNA levels. In the case–control study, 92 RCC patients and 580 cancer-free patients matched by age and gender were recruited. The apurinic/APE genotyping work was conducted with typical restriction fragment length polymorphism methodology after polymerase chain reaction. At the meanwhile, thirty renal tissue samples with variant genotypes were examined for their apurinic/APE mRNA and protein expressions by real-time quantitative reverse transcription method and Western blotting. The results showed that compared with the wild-type TT genotype, the people with TG and GG genotypes of apurinic/APE Asp148Glu had 0.88- and 1.09-fold risk of RCC, respectively. We have also examined the in vivo transcriptional (RNA) and translational (protein) levels with renal tissues of various apurinic/APE Asp148Glu genotypes, revealing that the apurinic/APE mRNA and protein were of similar levels among people of TT, TG, or GG genotypes. There was no joint gene-environment effect of apurinic/APE Asp148Glu genotype and smoking habit on RCC risk. The evidence indicated that apurinic/APE Asp148Glu genotypic variants did not alter its mRNA and protein expression among RCC patients. The genotype of apurinic/APE Asp148Glu may not serve as a proper predictive marker for RCC risk in Taiwan.

Despite significant achievements in the elucidation of the nature of protein-DNA contacts that control the specificity of nucleotide incision repair (NIR) by apurinic/apyrimidinic (AP) endonucleases, the question on how a given nucleotide is accommodated by the active site of the enzyme remains unanswered. Therefore, the main purpose of our study was to compare kinetics of conformational changes of three homologous APE1-like endonucleases (insect Drosophila melanogaster Rrp1, amphibian Xenopus laevis xAPE1, and fish Danio rerio zAPE1) during their interaction with various damaged DNA substrates, i.e., DNA containing an F-site (an uncleavable by DNA-glycosylases analog of an AP-site), 1,N6-ethenoadenosine (εA), 5,6-dihydrouridine (DHU), uridine (U), or the α-anomer of adenosine (αA). Pre-steady-state analysis of fluorescence time courses obtained for the interaction of the APE1-like enzymes with DNA substrates containing various lesions allowed us to outline a model of substrate recognition by this class of enzymes. It was found that the differences in rates of DNA substrates’ binding do not lead to significant differences in the cleavage efficiency of DNA containing a damaged base. The results suggest that the formation of enzyme–substrate complexes is not the key factor that limits enzyme turnover; the mechanisms of damage recognition and cleavage efficacy are related to fine conformational tuning inside the active site.

The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product–enzyme complex by the next enzyme in the pathway, resulting in the "passing-the-baton" model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1–DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme–product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polβ stimulates both i) APE1 binding to an abasic-site–containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polβ activities and suggest that Polβ is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER. • Interaction of fluorescently labeled APE1 with damaged DNA and Polβ was analyzed. • Labeled APE1 was sensitive to DNA binding, catalysis and release of cleaved product. • DNA-mediated coordination of APE1 and Polβ activities was analyzed by stopped-flow assay. • Polβ stimulates both APE1 binding to abasic DNA and its dissociation from product. [ABSTRACT FROM AUTHOR]

Human apurinic/apyrimidinic endonuclease APE1 catalyzes endonucleolytic hydrolysis of phosphodiester bonds on the 5′ side of structurally unrelated damaged nucleotides in DNA or native nucleotides in RNA. APE1 additionally possesses 3′-5′-exonuclease, 3′-phosphodiesterase, and 3′-phosphatase activities. According to structural data, endo- and exonucleolytic cleavage of DNA is executed in different complexes when the excised residue is everted from the duplex or placed within the intrahelical DNA cavity without nucleotide flipping. In this study, we investigated the functions of residues Arg177, Arg181, Tyr171 and His309 in the APE1 endo- and exonucleolytic reactions. The interaction between residues Arg177 and Met270, which was hypothesized recently to be a switch for endo- and exonucleolytic catalytic mode regulation, was verified by pre–steady-state kinetic analysis of the R177A APE1 mutant. The function of another DNA-binding–site residue, Arg181, was analyzed too; it changed its conformation when enzyme–substrate and enzyme–product complexes were compared. Mutation R181A significantly facilitated the product dissociation stage and only weakly affected DNA-binding affinity. Moreover, R181A reduced the catalytic rate constant severalfold due to a loss of contact with a phosphate group. Finally, the protonation/deprotonation state of residues Tyr171 and His309 in the catalytic reaction was verified by their substitution. Mutations Y171F and H309A inhibited the chemical step of the AP endonucleolytic reaction by several orders of magnitude with retention of capacity for (2 R ,3 S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran-containing-DNA binding and without changes in the pH dependence profile of AP endonuclease activity, indicating that deprotonation of these residues is likely not important for the catalytic reaction. • Endo- and exonuclease cleavage of a DNA by mutant forms of human APE1 was analyzed. • Process of protein-substrate interaction was analyzed by pre-steady state kinetics. • Conformational changes in APE1 and substrates were observed during their interactions. • The role of certain important amino acids during enzyme pathway was specified. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5′ side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron–electron double resonance (PELDOR, also known as DEER) spectroscopy and pre–steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2 R ,3 S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2′-deoxyuridine > α-anomer of 2′-deoxyadenosine >2′-deoxyuridine > undamaged DNA. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme–substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes. [Display omitted] • The molecular origin of broad substrate specificity of apurinic/apyrimidinic endonucleases is still not clear. • DEER spectroscopy and pre-steady-state kinetics allowed to elucidate DNA structural factors controlling enzyme specificity. • Recognition of lesion depends on the ability of a damaged nucleotide to change enzyme-induced DNA-bending and DNA-unwinding. • Common strategy of damage recognition by AP endonucleases from different structural families is announced for the first time. [ABSTRACT FROM AUTHOR]

Cellular oxidative stress from effects of reactive oxygen species (ROS) and its induced molecular response play an important role in the pathogenesis of various diseases and important biological processes. Nanostructure-based fluorescent probes can simultaneously map the localization and abundance of ROS and its induced molecules in living cells. However, proximity-induced fluorophore interaction leads to low sensitivity and poor specificity. Here, we engineered intramolecular Orthogonal Reporters (iOR) based on DNA nanostructure scaffolds. Fluorescence reporters in iOR were precisely located with appropriate distance by simulation eliminating the undesired interaction of fluorophores. iOR functionalized with nuclear localization signal peptide is capable of imaging ROS and its related DNA repair enzyme (APE1) in the nucleus and cytoplasm with high sensitivity and specificity. iOR discloses the strong positive relation of ROS and APE1 as well as their synergistic regulation in living cells and tumor-bearing mice. This fact is first presented by direct fluorescence imaging. This work reported a new procedure for developing nanostructure-based molecular probes and would find broad applications in cellular biology and nanotechnology. [Display omitted] • Fluorescence probes were integrated within DNA nanostructures guided by simulation tool eliminating crosstalk. • ROS and APE1 were quantitatively mapped in living cells and tumor bearing mice with spatiotemporal accuracy. • Elevated ROS level induces the overexpression of APE1 mainly because of the cumulative damaged bases in DNA. • This work demonstrates a generalizable strategy to construct nanostructure-based probes for imaging multiple targets. [ABSTRACT FROM AUTHOR]

Cytoplasmic localization of apurinic/apyrimidinic endonuclease 1 (APE1) correlates with different tumorigenic processes and poor prognosis in several cancer types. However, rare investigation into the prognosis value of cytoplasmic localization of APE1 was provided in ovarian cancer. The present study examined for the first time the cytoplasmic localization of APE1 in epithelial ovarian cancer (EOC) by immunohistochemistry. The relationship between cytoplasmic localization of APE1 and clinicopathological parameters, as well as the correlation between cytoplasmic localization of APE1 and prognosis, was investigated. We found that cytoplasmic positivity was significantly higher in EOCs with low tumor differentiation ( P = 0.002) and was significantly higher in advanced Federation International of Gynecology and Obstetrics (FIGO) stage (III + IV) patients compared to that in early FIGO stage (I + II) patients (40.7% vs. 11.8%; P = 0.002). No significant difference was observed in APE1 pattern referring to age, tumor size, family history, histological type, ascites, and lymphatic metastasis ( P > 0.05). In addition, a lower survival rate was found in patients with cytoplasmic positive localization of APE1 compared to that in patients with cytoplasmic negative localization ( P < 0.05). All these findings suggest that cytoplasmic localization of APE1 is associated with tumor progression and might be a valuable prognostic marker for EOC. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic endonuclease 1 (APE1 or Ref-1) is the major enzyme in mammals for processing abasic sites in DNA. These cytotoxic and mutagenic lesions arise via spontaneous rupture of the base-sugar bond or the removal of damaged bases by a DNA glycosylase. APE1 cleaves the DNA backbone 5′ to an abasic site, giving a 3′-OH primer for repair synthesis, and mediates other key repair activities. The DNA repair functions are essential for embryogenesis and cell viability. APE1-deficient cells are hypersensitive to DNA-damaging agents, and APE1 is considered an attractive target for inhibitors that could potentially enhance the efficacy of some anti-cancer agents. To enable an important new method for studying the structure, dynamics, catalytic mechanism, and inhibition of APE1, we assigned the chemical shifts (backbone and 13Cβ) of APE1 residues 39-318. We also report a protocol for refolding APE1, which was essential for achieving complete exchange of backbone amide sites for the perdeuterated protein. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP)-endonucleases are multifunctional enzymes that are required for cell viability. AP-endonucleases incise DNA 5′ to an AP-site; can recognize and process some damaged nucleosides; and possess 3′-phosphodiesterase, 3′-phosphatase, and endoribonuclease activities. To elucidate the mechanism of substrate cleavage in detail, we analyzed the effect of mono- and divalent metal ions on the exo- and endonuclease activities of four homologous APE1-like endonucleases (from an insect (Rrp1), amphibian (xAPE1), fish (zAPE1), and from humans (hAPE1)). It was found that the enzymes had similar patterns of dependence on metal ions' concentrations in terms of AP-endonuclease activity, suggesting that the main biological function (AP-site cleavage) was highly conserved among evolutionarily distant species. The efficiency of the 3′-5′ exonuclease activity was the highest in hAPE1 among these enzymes. In contrast, the endoribonuclease activity of the enzymes could be ranked as hAPE1 ≈ zAPE1 ≤ xAPE1 ≤ Rrp1. Taken together, the results revealed that the tested enzymes differed significantly in their capacity for substrate cleavage, even though the most important catalytic and substrate-binding amino acid residues were conserved. It can be concluded that substrate specificity and cleavage efficiency were controlled by factors external to the catalytic site, e.g., the N-terminal domain of these enzymes. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic endonuclease (APE1), a bifunctional AP endonuclease/redox factor, is important in DNA repair and redox signaling, may be associated with radioresistance. Here we investigate whether targeted inhibition of APE1 can sensitize tumor cells to irradiation in vitro and in vivo. We first constructed chimeric adenoviral vector Ad5/F35 carrying human APE1 siRNA (Ad5/F35-APE1 siRNA). The infectivity of chimeric Ad5/F35 to LOVO colon cancer cells was greater than that of Ad5. APE1 was strongly expressed and nuclear factor κB (NF-κB), a downstream molecule of APE1, known as a radioresistance factor, was constitutively active in LOVO cells. Infection of LOVO cells with Ad5/F35-APE1 siRNA resulted in a dose-dependent decrease of APE1 protein and AP endonuclease activity in vitro. Ad5/F35-APE1 siRNA significantly enhanced sensitivity of LOVO cells to irradiation in clonogenic survival assays, associated with increased cell apoptosis. The APE1 expression in LOVO cells was induced by irradiation in a dose-dependent manner, accompanied with the enhancement of DNA-binding activity of NF-κB and Ad5/F35-APE1 siRNA effectively inhibited constitutive and irradiation-induced APE1 expression and NF-κB activation. In a subcutaneous nude mouse colon cancer model, Ad5/F35-APE1 siRNA (5 × 108 IU, intratumoral injection) inhibited the expression of APE1 protein in LOVO xenografts, and significantly enhanced inhibition of tumor growth by irradiation. In conclusion, APE1 may be involved as one of the radioresistance factors, and targeted inhibition of APE1 shows an effective means of enhancing tumor sensitivity to radiotherapy.Cancer Gene Therapy (2008) 15, 625–635; doi:10.1038/cgt.2008.30; published online 6 June 2008 [ABSTRACT FROM AUTHOR]

The multifunctional DNA repair enzymes apurinic/apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3′-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5′ of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3′-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing α-anomeric 2′-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions. [ABSTRACT FROM AUTHOR]

Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to cancer risk. However, epidemiologic findings have been inconsistent. The authors conducted meta-analyses of associations between genes in the base excision repair pathway and cancer risk, focusing on three key genes: 8-oxoguanine DNA glycosylase (OGG1), apurinic/apyrimidinic endonuclease (APE1/APEX1), and x-ray repair cross-complementing group 1 (XRCC1). They found increased lung cancer risk among subjects carrying the OGG1 Cys/Cys genotype (odds ratio (OR) = 1.24, 95% confidence interval (CI): 1.01, 1.53), using 3,253 cases and 3,371 controls from seven studies; this is consistent with experimental evidence that this isoform exhibits decreased activity. They found a protective effect of the XRCC1 194Trp allele for tobacco-related cancers (OR = 0.86, 95% CI: 0.77, 0.95), using 4,895 cases and 5,977 controls from 16 studies; this is compatible with evidence of lower mutagen sensitivity for this allele. The XRCC1 399Gln/399Gln genotype was associated with increased risk of tobacco-related cancers among light smokers (OR = 1.38, 95% CI: 0.99, 1.94) but decreased risk among heavy smokers (OR = 0.71, 95% CI: 0.51, 0.99), suggesting effect modification by tobacco smoking. There was no association between cancer risk and the APE1/APEX1 Asp148Glu and XRCC1 Arg280His polymorphisms. Recommendations for future studies include pooling of individual data to facilitate evaluation of multigenic effects and detailed analysis of effect modification by environmental exposure. [ABSTRACT FROM AUTHOR]

Multifunctional mammalian apurinic/apyrimidinic endonuclease (APE, also known as redox factor-1; Ref-1) repairs baseless sites of damaged DNA caused by oxidative stress and regulates the redox state of various DNA binding proteins. Here, we examined the expression of APE/Ref-1 m-RNA in the mouse brain by in situ hybridization. We detected APE/Ref-1 transcripts throughout the mouse brain particularly in the clock oscillating neurons of the suprachiasmatic nucleus (SCN), hippocampal pyramidal cells, granular cells, and in monoaminergic neurons. In the circadian center SCN, levels of APE/ref-1 mRNA transcripts were constantly high, and were not influenced by either circadian rhythms or by exposure to light. [Copyright &y& Elsevier]

The 3′→5′-exonucleolytic activity of human apurinic/apyrimidinic endonuclease 1 (APE1) on mispaired DNA at the 3′-termini of recessed, nicked or gapped DNA molecules was analyzed and compared with the primary endonucleolytic activity. We found that under reaction conditions optimal for AP endonuclease activity the 3′→5′-exonuclease activity of APE1 manifests only at enzyme concentration elevated by 6–7 orders of magnitude. This activity does not show a preference to mismatched compared to matched DNA structures as well as to nicked or gapped DNA substrates in comparison to recessed ones. Therefore, the 3′→5′-exonuclease activity associated with APE1 can hardly be considered as key mechanism that improves fidelity of DNA repair. [Copyright &y& Elsevier]

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is ∼3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5′- and 3′-terminal untranslated regions and, at its 3′-terminus, SjR2 bears a short (TGAC)3 repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 from the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that ∼10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5′-untranslated region of SjR2 was >80% identical to the 3′-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni. [Copyright &y& Elsevier]

Proliferative diabetic retinopathy (PDR), neovascular age-related macular degeneration (nvAMD), retinopathy of prematurity (ROP) and other eye diseases are characterized by retinal and/or choroidal neovascularization, ultimately causing vision loss in millions of people worldwide. nvAMD and PDR are associated with aging and the number of those affected is expected to increase as the global median age and life expectancy continue to rise. With this increase in prevalence, the development of novel, orally bioavailable therapies for neovascular eye diseases that target multiple pathways is critical, since current anti-vascular endothelial growth factor (VEGF) treatments, delivered by intravitreal injection, are accompanied with tachyphylaxis, a high treatment burden and risk of complications. One potential target is apurinic/apyrimidinic endonuclease 1/reduction-oxidation factor 1 (APE1/Ref-1). The multifunctional protein APE1/Ref-1 may be targeted via inhibitors of its redox-regulating transcription factor activation activity to modulate angiogenesis, inflammation, oxidative stress response and cell cycle in neovascular eye disease; these inhibitors also have neuroprotective effects in other tissues. An APE1/Ref-1 small molecule inhibitor is already in clinical trials for cancer, PDR and diabetic macular edema. Efforts to develop further inhibitors are underway. APE1/Ref-1 is a novel candidate for therapeutically targeting neovascular eye diseases and alleviating the burden associated with anti-VEGF intravitreal injections. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes' intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding. [ABSTRACT FROM AUTHOR]

In the realm of DNA repair, base excision repair (BER) protein, APE1/Ref-1 (Apurinic/Apyrimidinic Endonuclease 1/Redox Effector - 1, also called APE1) has been studied for decades. However, over the past decade, APE1 has been established as a key player in reduction-oxidation (redox) signaling. In the review by Caston et al. ( The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease), multiple roles of APE1 in cancer and other diseases are summarized. In this Review, we aim to expand on the contributions of APE1 to various diseases and its effect on disease progression. In the scope of cancer, more recent roles for APE1 have been identified in cancer cell metabolism, as well as chemotherapy-induced peripheral neuropathy (CIPN) and inflammation. Outside of cancer, APE1 signaling may be a critical factor in inflammatory bowel disease (IBD) and is also an emergent area of investigation in retinal ocular diseases. The ability of APE1 to regulate multiple transcription factors (TFs) and therefore multiple pathways that have implications outside of cancer, makes it a particularly unique and enticing target. We discuss APE1 redox inhibitors as a means of studying and potentially combating these diseases. Lastly, we examine the role of APE1 in RNA metabolism. Overall, this article builds on our previous review to elaborate on the roles and conceivable regulation of important pathways by APE1 in multiple diseases., Competing Interests: Conflict of Interest MRK is a member of the Ocuphire medical advisory board and CSO and co-founder of Apexian Pharmaceuticals which licensed APX3330 and other APX compounds from Indiana University School of Medicine. Ocuphire Pharma licensed APX3330 for ocular and diabetes indications from Apexian Pharmaceuticals.

Transcripts of many enzymes involved in base excision repair (BER) undergo extensive alternative splicing, but functions of the corresponding alternative splice variants remain largely unexplored. In this review, we cover the studies describing the common alternatively spliced isoforms and disease-associated variants of DNA glycosylases, AP-endonuclease 1, and DNA polymerase beta. We also discuss the roles of alternative splicing in the regulation of their expression, catalytic activities, and intracellular transport. [ABSTRACT FROM AUTHOR]

Background: The thioredoxin system maintains redox balance through the action of thioredoxin and thioredoxin reductase. Thioredoxin regulates the activity of various substrates, including those that function to counteract cellular oxidative stress. These include the peroxiredoxins, methionine sulfoxide reductase A and specific transcription factors. Of particular relevance is Redox Factor-1, which in turn activates other redox-regulated transcription factors., Scope of Review: Experimentally defined transcription factor binding sites in the human thioredoxin and thioredoxin reductase gene promoters together with promoters of the major thioredoxin system substrates involved in regulating cellular redox status are discussed. An in silico approach was used to identify potential putative binding sites for these transcription factors in all of these promoters., Major Conclusions: Our analysis reveals that many redox gene promoters contain the same transcription factor binding sites. Several of these transcription factors are in turn redox regulated. The ARE is present in several of these promoters and is bound by Nrf2 during various oxidative stress stimuli to upregulate gene expression. Other transcription factors also bind to these promoters during the same oxidative stress stimuli, with this redundancy supporting the importance of the antioxidant response. Putative transcription factor sites were identified in silico, which in combination with specific regulatory knowledge for that gene promoter may inform future experiments., General Significance: Redox proteins are involved in many cellular signalling pathways and aberrant expression can lead to disease or other pathological conditions. Therefore understanding how their expression is regulated is relevant for developing therapeutic agents that target these pathways., (© 2013.)

Increased germ cell apoptosis is related to oxidative DNA damage; therefore, we investigated whether there was a significant change in apurinic/apyrimidinic endonuclease (APE) in varicoceles. Experimental varicoceles were created by partial ligation of the left renal vein of adult male Sprague-Dawley rats, which were sacrificed at 1, 3 and 6 weeks after varicocele creation. Testicular tissues were sampled for TUNEL, Western blotting and immunohistochemistry. There was a significant increase in apoptotic germ cells in the ipsilateral testes 6 weeks after varicocele creation. Increased activation of p53, Bax and cleaved caspase-3 in the left testes was also noted. APE increased activation until 3 weeks after varicocele creation, and then decreased at 6 weeks after varicocele surgery. The spermatocytes were immunostained for both 8-hydroxy-2'-deoxyguanosine and APE, but the spermatogonia revealed only APE immunopositivity in the defective tubules. These results suggest that repression of APE is an underlying mechanism of augmented p53-dependent apoptosis in varicocele-induced rat testes and that remaining APE in the spermatogonia plays a decisive role in regaining testicular spermatogenic function after varicocelectomy., (Copyright © 2013 Elsevier GmbH. All rights reserved.)

A full-atomic molecular model of human apurinic/apyrimidinic endonuclease APE1, an important enzyme in the DNA repair system, has been constructed. The research consisted of hybrid quantum mechanics/molecular mechanics modeling of the enzyme-substrate interactions, as well as calculations of the ionization states of the amino acid residues of the active site of the enzyme. The choice of the APE1 mechanism with an Asp210 residue as a proton acceptor was validated by means of a generalization of modeling and experimental data. Interactions were revealed in the active site that are of greatest significance for binding the substrate and potential APE1 inhibitors (potential co-drugs of interest in the chemo- and radiotherapy of oncological diseases).