Nanoscale Interaction of Endonuclease APE1 with DNA.

Apurinic/apyrimidinic endonuclease 1 (APE1) is involved in DNA repair and transcriptional regulation mechanisms. This multifunctional activity of APE1 should be supported by specific structural properties of APE1 that have not yet been elucidated. Herein, we applied atomic force microscopy (AFM) to characterize the interactions of APE1 with DNA containing two well-separated G-rich segments. Complexes of APE1 with DNA containing G-rich segments were visualized, and analysis of the complexes revealed the affinity of APE1 to G-rich DNA sequences, and their yield was as high as 53%. Furthermore, APE1 is capable of binding two DNA segments leading to the formation of loops in the DNA-APE1 complexes. The analysis of looped APE1-DNA complexes revealed that APE1 can bridge G-rich segments of DNA. The yield of loops bridging two G-rich DNA segments was 41%. Analysis of protein size in various complexes was performed, and these data showed that loops are formed by APE1 monomer, suggesting that APE1 has two DNA binding sites. The data led us to a model for the interaction of APE1 with DNA and the search for the specific sites. The implication of these new APE1 properties in organizing DNA, by bringing two distant sites together, for facilitating the scanning for damage and coordinating repair and transcription is discussed.

Thermococcus kodakarensis TK0353 is a novel AP lyase with a new fold.

Hyperthermophilic organisms thrive in extreme environments prone to high levels of DNA damage. Growth at high temperature stimulates DNA base hydrolysis resulting in apurinic/apyrimidinic (AP) sites that destabilize the genome. Organisms across all domains have evolved enzymes to recognize and repair AP sites to maintain genome stability. The hyperthermophilic archaeon Thermococcus kodakarensis encodes several enzymes to repair AP site damage including the essential AP endonuclease TK endonuclease IV. Recently, using functional genomic screening, we discovered a new family of AP lyases typified by TK0353. Here, using biochemistry, structural analysis, and genetic deletion, we have characterized the TK0353 structure and function. TK0353 lacks glycosylase activity on a variety of damaged bases and is therefore either a monofunctional AP lyase or may be a glycosylase-lyase on a yet unidentified substrate. The crystal structure of TK0353 revealed a novel fold, which does not resemble other known DNA repair enzymes. The TK0353 gene is not essential for T. kodakarensis viability presumably because of redundant base excision repair enzymes involved in AP site processing. In summary, TK0353 is a novel AP lyase unique to hyperthermophiles that provides redundant repair activity necessary for genome maintenance.

Structural and biochemical insights into NEIL2's preference for abasic sites.

Cellular DNA is subject to damage from a multitude of sources and repair or bypass of sites of damage utilize an array of context or cell cycle dependent systems. The recognition and removal of oxidatively damaged bases is the task of DNA glycosylases from the base excision repair pathway utilizing two structural families that excise base lesions in a wide range of DNA contexts including duplex, single-stranded and bubble structures arising during transcription. The mammalian NEIL2 glycosylase of the Fpg/Nei family excises lesions from each of these DNA contexts favoring the latter two with a preference for oxidized cytosine products and abasic sites. We have determined the first liganded crystal structure of mammalian NEIL2 in complex with an abasic site analog containing DNA duplex at 2.08 Å resolution. Comparison to the unliganded structure revealed a large interdomain conformational shift upon binding the DNA substrate accompanied by local conformational changes in the C-terminal domain zinc finger and N-terminal domain void-filling loop necessary to position the enzyme on the DNA. The detailed biochemical analysis of NEIL2 with an array of oxidized base lesions indicates a significant preference for its lyase activity likely to be paramount when interpreting the biological consequences of variants.

PARP1 Incises Abasic Sites and Covalently Cross-links to 3'-DNA Termini via Cysteine Addition Not Reductive Amination.

Poly [ADP-ribose] polymerase 1 (PARP1) is a ubiquitous nuclear enzyme that plays multifaceted roles in the cellular response to DNA damage. Previous studies demonstrated that PARP1 incises the most frequently formed DNA lesion, the apurinic/apyrimidinic (AP) site, and in the process is trapped as a DNA-PARP1 cross-link at the 3'-terminus. The covalent linkage was proposed to be composed of a secondary amine resulting from formal reductive amination of an initially formed incision product. PARP1 cysteine residues were proposed to reduce the initially formed Schiff base. Here, we report evidence to support a different mechanism in which DNA-PARP1 cross-links result from cysteine addition to incised AP sites.

AUF1 Recognizes 8-Oxo-Guanosine Embedded in DNA and Stimulates APE1 Endoribonuclease Activity.

Aims: The existence of modified ribonucleotide monophosphates embedded in genomic DNA, as a consequence of oxidative stress conditions, including 8-oxo-guanosine and ribose monophosphate abasic site (rAP), has been recently highlighted by several works and associated with oxidative stress conditions. Although human apurinic-apyrimidinic endodeoxyribonuclease 1 (APE1), a key enzyme of the base-excision repair pathway, repairs rAP sites and canonical deoxyribose monophosphate abasic sites with similar efficiency, its incision-repairing activity on 8-oxo-guanosine is very weak. The aims of this work were to: (i) identify proteins able to specifically bind 8-oxo-guanosine embedded in DNA and promote APE1 endoribonuclease activity on this lesion, and (ii) characterize the molecular and biological relevance of this interaction using human cancer cell lines. Results: By using an unbiased proteomic approach, we discovered that the AU-rich element RNA-binding protein 1 (AUF1) actively recognizes 8-oxo-guanosine and stimulates the APE1 enzymatic activity on this DNA lesion. By using orthogonal approaches, we found that: (i) the interaction between AUF1 and APE1 is modulated by H2O2-treatment; (ii) depletion of APE1 and AUF1 causes the accumulation of single- and double- strand breaks; and (iii) both proteins are involved in modulating the formation of DNA:RNA hybrids. Innovation: These results establish unexpected functions of AUF1 in modulating genome stability and improve our knowledge of APE1 biology with respect to 8-oxo-guanosine embedded in DNA. Conclusion: By showing a novel function of AUF1, our findings shed new light on the process of genome stability in mammalian cells toward oxidative stress-related damages. Antioxid. Redox Signal. 39, 411-431.

Characterizing inhibitors of human AP endonuclease 1.

AP endonuclease 1 (APE1) processes DNA lesions including apurinic/apyrimidinic sites and 3´-blocking groups, mediating base excision repair and single strand break repair. Much effort has focused on developing specific inhibitors of APE1, which could have important applications in basic research and potentially lead to clinical anticancer agents. We used structural, biophysical, and biochemical methods to characterize several reported inhibitors, including 7-nitroindole-2-carboxylic acid (CRT0044876), given its small size, reported potency, and widespread use for studying APE1. Intriguingly, NMR chemical shift perturbation (CSP) experiments show that CRT0044876 and three similar indole-2-carboxylic acids bind a pocket distal from the APE1 active site. A crystal structure confirms these findings and defines the pose for 5-nitroindole-2-carboxylic acid. However, dynamic light scattering experiments show the indole compounds form colloidal aggregates that could bind (sequester) APE1, causing nonspecific inhibition. Endonuclease assays show the compounds lack significant APE1 inhibition under conditions (detergent) that disrupt aggregation. Thus, binding of the indole-2-carboxylic acids at the remote pocket does not inhibit APE1 repair activity. Myricetin also forms aggregates and lacks APE1 inhibition under aggregate-disrupting conditions. Two other reported compounds (MLS000552981, MLS000419194) inhibit APE1 in vitro with low micromolar IC50 and do not appear to aggregate in this concentration range. However, NMR CSP experiments indicate the compounds do not bind specifically to apo- or Mg2+-bound APE1, pointing to a non-specific mode of inhibition, possibly DNA binding. Our results highlight methods for rigorous interrogation of putative APE1 inhibitors and should facilitate future efforts to discover compounds that specifically inhibit this important repair enzyme.

Regulation of futile ligation during early steps of BER in M. tuberculosis is carried out by a ß-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 Mtbß-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 Mtbß-clamp and MtbLigA. The studies support that in the apo-complex MtbXthA binds subsite-I of Mtbß-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 Mtbß-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.

The Impact of DFT Functional, Cluster Model Size, and Implicit Solvation on the Structural Description of Single-Metal-Mediated DNA Phosphodiester Bond Cleavage: The Case Study of APE1.

Phosphodiester bond hydrolysis in nucleic acids is a ubiquitous reaction that can be facilitated by enzymes called nucleases, which often use metal ions to achieve catalytic function. While a two-metal-mediated pathway has been well established for many enzymes, there is growing support that some enzymes require only one metal for the catalytic step. Using human apurinic/apyrimidinic endonuclease (APE1) as a prototypical example and cluster models, this study clarifies the impact of DFT functional, cluster model size, and implicit solvation on single-metal-mediated phosphodiester bond cleavage and provides insight into how to efficiently model this chemistry. Initially, a model containing 69 atoms built from a high-resolution X-ray crystal structure is used to explore the reaction pathway mapped by a range of DFT functionals and basis sets, which provides support for the use of standard functionals (M06-2X and B3LYP-D3) to study this reaction. Subsequently, systematically increasing the model size to 185 atoms by including additional amino acids and altering residue truncation points highlights that small models containing only a few amino acids or ß carbon truncation points introduce model strains and lead to incorrect metal coordination. Indeed, a model that contains all key residues (general base and acid, residues that stabilize the substrate, and amino acids that maintain the metal coordination) is required for an accurate structural depiction of the one-metal-mediated phosphodiester bond hydrolysis by APE1, which results in 185 atoms. The additional inclusion of the broader enzyme environment through continuum solvation models has negligible effects. The insights gained in the present work can be used to direct future computational studies of other one-metal-dependent nucleases to provide a greater understanding of how nature achieves this difficult chemistry.

Structures of topoisomerase V in complex with DNA reveal unusual DNA-binding mode and novel relaxation mechanism.

Topoisomerase V is a unique topoisomerase that combines DNA repair and topoisomerase activities. The enzyme has an unusual arrangement, with a small topoisomerase domain followed by 12 tandem (HhH)2 domains, which include 3 AP lyase repair domains. The uncommon architecture of this enzyme bears no resemblance to any other known topoisomerase. Here, we present structures of topoisomerase V in complex with DNA. The structures show that the (HhH)2 domains wrap around the DNA and in this manner appear to act as a processivity factor. There is a conformational change in the protein to expose the topoisomerase active site. The DNA bends sharply to enter the active site, which melts the DNA and probably facilitates relaxation. The structures show a DNA-binding mode not observed before and provide information on the way this atypical topoisomerase relaxes DNA. In common with type IB enzymes, topoisomerase V relaxes DNA using a controlled rotation mechanism, but the structures show that topoisomerase V accomplishes this in different manner. Overall, the structures firmly establish that type IC topoisomerases form a distinct type of topoisomerases, with no similarities to other types at the sequence, structural, or mechanistic level. They represent a completely different solution to DNA relaxation.

Cysteine Oxidation to Sulfenic Acid in APE1 Aids G-Quadruplex Binding While Compromising DNA Repair.

Apurinic/apyrimidinic endonuclease-1 (APE1) is a base excision repair (BER) enzyme that is also engaged in transcriptional regulation. Previous work demonstrated that the enzymatic stalling of APE1 on a promoter G-quadruplex (G4) recruits transcription factors during oxidative stress for gene regulation. Also, during oxidative stress, cysteine (Cys) oxidation is a post-translational modification (PTM) that can change a protein's function. The current study provides a quantitative survey of cysteine oxidation to sulfenic acid in APE1 and how this PTM at specific cysteine residues affects the function of APE1 toward the NEIL3 gene promoter G4 bearing an abasic site. Of the seven cysteine residues in APE1, five (C65, C93, C208, C296, and C310) were prone to carbonate radical anion oxidation to yield sulfenic acids that were identified and quantified by mass spectrometry. Accordingly, five Cys-to-serine (Ser) mutants of APE1 were prepared and found to have attenuated levels of endonuclease activity, depending on the position, while KD values generally decreased for G4 binding, indicating greater affinity. These data support the concept that cysteine oxidation to sulfenic acid can result in modified APE1 that enhances G4 binding at the expense of endonuclease activity during oxidative stress. Cysteine oxidation to sulfenic acid residues should be considered as one of the factors that may trigger a switch from base excision repair activity to transcriptional modulation by APE1.

Pre-steady-state kinetic and mutational insights into mechanisms of endo- and exonuclease DNA processing by mutant forms of human AP endonuclease.

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 (2R,3S)-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.

Research Progress in Human AP Endonuclease 1: Structure, Catalytic Mechanism, and Inhibitors.

The high stability of phosphodiester bonds is considered to be one of the important reasons for the genetic role of nucleic acids, and their cleavage is also the core of many key biochemical processes, including DNA replication/ repair, and RNA processing/ degradation. As an important part of the base excision repair (BER) pathway, human apurinic/ apyrimidinic endonuclease 1 (APE1) is indispensable for the repair of abasic sites and other DNA damage, including ionizing radiation, DNA covalently bonding induced by cytotoxic antitumor drugs, etc. For tumor cells, the DNA repair activity of APE1 may lead to the occurrence of radiotherapy and chemotherapy resistance. The overexpression of APE1 often poses a serious threat to the effectiveness of tumor treatment, indicating a longer time, a much larger dose, less effective chemotherapy, and poor prognosis. It is of great urgency to design novel APE1 inhibitors. Rational design and modification of inhibitor molecules are closely related to the research progress of both structural biology and catalytic mechanism. In this review, the structure, catalytic mechanism, inhibitors, and other important biochemical information regarding APE1 are summarized, which will help in the design and modification of drug molecules targeting APE1.

Dynamics and Conformational Changes in Human NEIL2 DNA Glycosylase Analyzed by Hydrogen/Deuterium Exchange Mass Spectrometry.

Base excision DNA repair (BER) is necessary for removal of damaged nucleobases from the genome and their replacement with normal nucleobases. BER is initiated by DNA glycosylases, the enzymes that cleave the N-glycosidic bonds of damaged deoxynucleotides. Human endonuclease VIII-like protein 2 (hNEIL2), belonging to the helix-two-turn-helix structural superfamily of DNA glycosylases, is an enzyme uniquely specific for oxidized pyrimidines in non-canonical DNA substrates such as bubbles and loops. The structure of hNEIL2 has not been solved; its closest homologs with known structures are NEIL2 from opossum and from giant mimivirus. Here we analyze the conformational dynamics of free hNEIL2 using a combination of hydrogen/deuterium exchange mass spectrometry, homology modeling and molecular dynamics simulations. We show that a prominent feature of vertebrate NEIL2 - a large insert in its N-terminal domain absent from other DNA glycosylases - is unstructured in solution. It was suggested that helix-two-turn-helix DNA glycosylases undergo open-close transition upon DNA binding, with the large movement of their N- and C-terminal domains, but the open conformation has been elusive to capture. Our data point to the open conformation as favorable for free hNEIL2 in solution. Overall, our results are consistent with the view of hNEIL2 as a conformationally flexible protein, which may be due to its participation in the repair of non-canonical DNA structures and/or to the involvement in functional and regulatory protein-protein interactions.

Differential repair enzyme-substrate selection within dynamic DNA energy landscapes.

We demonstrate that reshaping of the dynamic, bulged-loop energy landscape of DNA triplet repeat ensembles by the presence of an abasic site alters repair outcomes by the APE1 enzyme. This phenomenon depends on the structural context of the lesion, despite the abasic site always having the same neighbors in sequence space. We employ this lesion-induced redistribution of DNA states and a kinetic trap to monitor different occupancies of the DNA bulge loop states. We show how such dynamic redistribution and associated differential occupancies of DNA states impact APE1 repair outcomes and APE1 induced interconversions. We correlate the differential biophysical properties of the dynamic, DNA ensemble states, with their ability to be recognized and processed as substrates by the APE1 DNA repair enzyme. Enzymatic digestions and biophysical characterizations reveal that APE1 cuts a fraction (10-12%) of the dynamic, rollameric substrates within the initial kinetic distribution. APE1 interactions also 'induce' rollamer redistribution from a kinetically trapped distribution to an equilibrium distribution, the latter not containing viable APE1 substrates. We distinguish between kinetically controlled ensemble (re)distributions of potential DNA substrates, versus thermodynamically controlled ensemble (re)distribution; features of importance to DNA regulation. We conclude that APE1 activity catalyzes/induces ensembles that represent the thermodynamically optimal loop distribution, yet which also are nonviable substrate states for abasic site cleavage by APE1. We propose that by inducing substrate redistributions in a dynamic energy landscape, the enzyme actually reduces the available substrate competent species for it to process, reflective of a regulatory mechanism for enzymatic self-repression. If this is a general phenomenon, such a consequence would have a profound impact on slowing down and/or misdirecting DNA repair within dynamic energy landscapes, as exemplified here within triplet repeat domains. In short, APE1-instigated redistribution of potential substrates induces a preferred pathway to an equilibrium ensemble of enzymatically incompetent states.

Inhibition of APE1/Ref-1 for Neovascular Eye Diseases: From Biology to Therapy.

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.

Structural dissection of sequence recognition and catalytic mechanism of human LINE-1 endonuclease.

Long interspersed nuclear element-1 (L1) is an autonomous non-LTR retrotransposon comprising ∼20% of the human genome. L1 self-propagation causes genomic instability and is strongly associated with aging, cancer and other diseases. The endonuclease domain of L1's ORFp2 protein (L1-EN) initiates de novo L1 integration by nicking the consensus sequence 5'-TTTTT/AA-3'. In contrast, related nucleases including structurally conserved apurinic/apyrimidinic endonuclease 1 (APE1) are non-sequence specific. To investigate mechanisms underlying sequence recognition and catalysis by L1-EN, we solved crystal structures of L1-EN complexed with DNA substrates. This showed that conformational properties of the preferred sequence drive L1-EN's sequence-specificity and catalysis. Unlike APE1, L1-EN does not bend the DNA helix, but rather causes 'compression' near the cleavage site. This provides multiple advantages for L1-EN's role in retrotransposition including facilitating use of the nicked poly-T DNA strand as a primer for reverse transcription. We also observed two alternative conformations of the scissile bond phosphate, which allowed us to model distinct conformations for a nucleophilic attack and a transition state that are likely applicable to the entire family of nucleases. This work adds to our mechanistic understanding of L1-EN and related nucleases and should facilitate development of L1-EN inhibitors as potential anticancer and antiaging therapeutics.

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.

Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases.

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.

Molecular Mechanisms Regulating the DNA Repair Protein APE1: A Focus on Its Flexible N-Terminal Tail Domain.

APE1 (DNA (apurinic/apyrimidinic site) endonuclease 1) is a key enzyme of one of the major DNA repair routes, the BER (base excision repair) pathway. APE1 fulfils additional functions, acting as a redox regulator of transcription factors and taking part in RNA metabolism. The mechanisms regulating APE1 are still being deciphered. Structurally, human APE1 consists of a well-characterized globular catalytic domain responsible for its endonuclease activity, preceded by a conformationally flexible N-terminal extension, acquired along evolution. This N-terminal tail appears to play a prominent role in the modulation of APE1 and probably in BER coordination. Thus, it is primarily involved in mediating APE1 localization, post-translational modifications, and protein-protein interactions, with all three factors jointly contributing to regulate the enzyme. In this review, recent insights on the regulatory role of the N-terminal region in several aspects of APE1 function are covered. In particular, interaction of this region with nucleophosmin (NPM1) might modulate certain APE1 activities, representing a paradigmatic example of the interconnection between various regulatory factors.

Apurinic/Apyrimidinic Endonuclease 2 (APE2): An ancillary enzyme for contextual base excision repair mechanisms to preserve genome stability.

The genome of living organisms frequently undergoes various types of modifications which are recognized and repaired by the relevant repair mechanisms. These repair pathways are increasingly being deciphered to understand the mechanisms. Base excision repair (BER) is indispensable to maintain genome stability. One of the enigmatic repair proteins of BER, Apurinic/Apyrimidinic Endonuclease 2 (APE2), like APE1, is truly multifunctional and demonstrates the independent and non-redundant function in maintaining the genome integrity. APE2 is involved in ATR-Chk1 mediated DNA damage response. It also resolves topoisomerase1 mediated cleavage complex intermediate which is formed while repairing misincorporated ribonucleotides in the absence of functional RNase H2 mediated excision repair pathway. BER participates in the demethylation pathway and the role of Arabidopsis thaliana APE2 is demonstrated in this process. Moreover, APE2 is synthetically lethal to BRCA1, BRCA2, and RNase H2, and its homolog, APE1 fails to complement the function. Hence, the role of APE2 is not just an alternate to the repair mechanisms but has implications in diverse functional pathways related to the maintenance of genome integrity. This review analyses genomic features of APE2 and delineates its enzyme function as error-prone as well as efficient and accurate repair protein based on the studies on mammalian or its homolog proteins from model systems such as Arabidopsis thaliana, Schizosaccharomyces pombe, Trypanosoma curzi, Xenopus laevis, Danio rerio, Mus musculus, and Homo sapiens.