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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]

Escherichia coli endonuclease VIII (Endo VIII) is a DNA glycosylase with substrate specificity for a wide range of oxidatively damaged pyrimidine bases. Endo VIII catalyzes hydrolysis of the N-glycosidic bond and β, δ-elimination of 3'- and 5'-phosphate groups of an apurinic/apyrimidinic site. Single mutants of Endo VIII L70S, L70W, Y71W, F121W, F230W, and P253W were analyzed here with the aim to elucidate the kinetic mechanism of protein conformational adjustment during damaged-nucleotide recognition and catalytic-complex formation. F121W substitution leads to a slight reduction of DNA binding and catalytic activity. F230W substitution slows the rate of the δ-elimination reaction indicating that interaction of Phe230 with a 5'-phosphate group proceeds in the latest catalytic step. P253W Endo VIII has the same activity as the wild type (WT) enzyme. Y71W substitution slightly reduces the catalytic activity due to the effect on the later steps of catalytic-complex formation. Both L70S and L70W substitutions significantly decrease the catalytic activity, indicating that Leu70 plays an important role in the course of enzyme-DNA catalytic complex formation. Our data suggest that Leu70 forms contacts with DNA earlier than Tyr71 does. Therefore, most likely, Leu70 plays the role of a DNA lesion "sensor", which is used by Endo VIII for recognition of a DNA damage site. [ABSTRACT FROM AUTHOR]

Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5′-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified. [ABSTRACT FROM AUTHOR]

Human apurinic/apyrimidinic (AP) endonuclease APE1 hydrolyzes phosphodiester bonds on the 5′ side of an AP-site, and some damaged nucleotides such as 1,N6-ethenoadenosine (εA), α-adenosine (αA), and 5,6-dihydrouridine (DHU). To investigate the mechanism behind the broad substrate specificity of APE1, we analyzed pre-steady-state kinetics of conformational changes in DNA and the enzyme during DNA binding and damage recognition. Molecular dynamics simulations of APE1 complexes with one of damaged DNA duplexes containing εA, αA, DHU, or an F-site (a stable analog of an AP-site) revealed the involvement of residues Asn229, Thr233, and Glu236 in the mechanism of DNA lesion recognition. The results suggested that processing of an AP-site proceeds faster in comparison with nucleotide incision repair substrates because eversion of a small abasic site and its insertion into the active site do not include any unfavorable interactions, whereas the insertion of any target nucleotide containing a damaged base into the APE1 active site is sterically hindered. Destabilization of the α-helix containing Thr233 and Glu236 via a loss of the interaction between these residues increased the plasticity of the damaged-nucleotide binding pocket and the ability to accommodate structurally different damaged nucleotides. Nonetheless, the optimal location of εA or αA in the binding pocket does not correspond to the optimal conformation of catalytic amino acid residues, thereby significantly decreasing the cleavage efficacy for these substrates. [ABSTRACT FROM AUTHOR]

Apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae is known as a key player of the base excision DNA repair (BER) pathway in yeast. BER is initiated by DNA glycosylases, whereas Apn1 can start DNA repair individually in the nucleotide incision repair (NIR) pathway. The aim of this research was to elucidate kinetic and structural dynamic aspects of Apn1 involvement in the NIR process. One of the key characteristics of AP endonuclease's interactions is known to be divalent metal ions playing a part of a cofactor. Well-studied human APE1 employs Mg 2+ ions, with metal ion concentration's affecting enzymatic activity exerted by APE1. In our study, we aimed to test the effect of the Mg 2+ ion on Apn1's NIR catalysis by examining structural dynamics of DNA during the interaction in real time using the stopped-flow technique. To test NIR activity of Apn1, deoxyribooligonucleotide duplexes containing a 5,6-dihydro-2′-deoxyuridine (DHU) residue were employed as substrates. A 2-aminopurine (2-aPu) residue was a reporter group fluorescence intensity of which was detected during Apn1–DNA interactions. NIR activity of both WT and H83A Apn1 was found to be arrested during the interaction with a DNA duplex containing the 2-aPu residue upstream of DHU. We conducted molecular dynamics simulations to elucidate the structural features of complexes of the enzyme with DHU-containing DNAs. The NIR recruiting S. cerevisiae Apn1 proceeds via multistep rearrangements of the complex of Apn1 with a DHU-containing DNA substrate and results in the incised product of the reaction. For wild-type Apn1, the catalytic rate constants do not depend on the Mg 2+ concentration, i.e., they are equal in NIR and BER buffers, with equilibrium association constant K a being 10-fold higher in NIR buffer. Our data reveal more delicate regulation of Apn1's NIR activity due to the more complicated kinetic mechanism, as compared to BER. [ABSTRACT FROM AUTHOR]

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3′-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl- N -purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1–DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme–substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. Significance Statement The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair. [ABSTRACT FROM AUTHOR]

Background The apurinic/apyrimidinic (AP) endonuclease Apn1 from Saccharomyces cerevisiae is a key enzyme involved in the base excision repair (BER) at the cleavage stage of abasic sites (AP sites) in DNA. The crystal structure of Apn1 from S. cerevisiae is unresolved. Based on its high amino acid homology to Escherichia coli Endo IV, His-83 is believed to coordinate one of three Zn 2 + ions in Apn1's active site similar to His-69 in Endo IV. Substituting His-83 with Ala is proposed to decrease the AP endonuclease activity of Apn1 owing to weak coordination of Zn 2 + ions involved in enzymatic catalysis. Methods The kinetics of recognition, binding, and incision of DNA substrates with the H83A Apn1 mutant was investigated. The stopped-flow method detecting fluorescence intensity changes of 2-aminopurine (2-aPu) was used to monitor the conformational dynamics of DNA at pre-steady-state conditions. Results We found substituting His-83 with Ala influenced catalytic complex formation and further incision of the damaged DNA strand. The H83A Apn1 catalysis depends not only on the location of the mismatch relative to the abasic site in DNA, but also on the nature of damage. Conclusions We consider His-83 properly coordinates the active site Zn 2 + ion playing a crucial role in catalytic incision stage. Our data prove suppressed enzymatic activity of H83A Apn1 results from the reduced number of active site Zn 2 + ions. General significance Our study provides insights into mechanistic specialty of AP site repair by yeast AP endonuclease Apn1 of Endo IV family, which members are not found in mammals, but are present in many microorganisms. The results will provide useful guidelines for design of new anti-fungal and anti-malarial agents. [ABSTRACT FROM AUTHOR]

Background DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Methods Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4 cat and UNG from different structural superfamilies were used. Results We found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. Conclusions We hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4 cat , AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. General significance Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. [ABSTRACT FROM AUTHOR]

Mammalian AP endonuclease 1 is a pivotal enzyme of the base excision repair pathway acting on apurinic/apyrimidinic sites. Previous structural and biochemical studies showed that the conserved Asn-212 residue is important for the enzymatic activity of APE1. Here, we report a comprehensive pre-steady-state kinetic analysis of two APE1 mutants, each containing amino acid substitutions at position 212, to ascertain the role of Asn-212 in individual steps of the APE1 catalytic mechanism. We applied the stopped-flow technique for detection of conformational transitions in the mutant proteins and DNA substrates during the catalytic cycle, using fluorophores that are sensitive to the micro-environment. Our data indicate that Asn-212 substitution by Asp reduces the rate of the incision step by ~550-fold, while Ala substitution results in ~70,000-fold decrease. Analysis of the binding steps revealed that both mutants continued to rapidly and efficiently bind to abasic DNA containing the natural AP site or its tetrahydrofuran analogue (F). Moreover, transient kinetic analysis showed that N212A APE1 possessed a higher binding rate and a higher affinity for specific substrates compared to N212D APE1. Molecular dynamics (MD) simulation revealed a significant dislocation of the key catalytic residues of both mutant proteins relative to wild-type APE1. The analysis of the model structure of N212D APE1 provides evidence for alternate hydrogen bonding between Asn-212 and Asp-210 residues, whereas N212A possesses an extended active site pocket due to Asn removal. Taken together, these biochemical and MD simulation results indicate that Asn-212 is essential for abasic DNA incision, but is not crucial for effective recognition/binding. [ABSTRACT FROM AUTHOR]

Abstract: Background: Extensive structural studies of human DNA glycosylase hOGG1 have revealed essential conformational changes of the enzyme. However, at present there is little information about the time scale of the rearrangements of the protein structure as well as the dynamic behavior of individual amino acids. Methods: Using pre-steady-state kinetic analysis with Trp and 2-aminopurine fluorescence detection the conformational dynamics of hOGG1 wild-type (WT) and mutants Y203W, Y203A, H270W, F45W, F319W and K249Q as well as DNA–substrates was examined. Results: The roles of catalytically important amino acids F45, Y203, K249, H270, and F319 in the hOGG1 enzymatic pathway and their involvement in the step-by-step mechanism of oxidative DNA lesion recognition and catalysis were elucidated. Conclusions: The results show that Tyr-203 participates in the initial steps of the lesion site recognition. The interaction of the His-270 residue with the oxoG base plays a key role in the insertion of the damaged base into the active site. Lys-249 participates not only in the catalytic stages but also in the processes of local duplex distortion and flipping out of the oxoG residue. Non-damaged DNA does not form a stable complex with hOGG1, although a complex with a flipped out guanine base can be formed transiently. General significance: The kinetic data obtained in this study significantly improves our understanding of the molecular mechanism of lesion recognition by hOGG1. [Copyright &y& Elsevier]

Abstract: Endonuclease VIII (Nei) from Escherichia coli is a DNA repair enzyme that removes a wide range of oxidized pyrimidine bases from DNA. As inferred from the crystal structures and biochemical studies, recognition of DNA lesions by Nei involves several conformational changes in both protein and DNA, such as DNA kinking, damaged base eversion into the enzyme''s active site, and insertion of a loop of the enzyme into the void formed by the eversion. Excision of the damaged base by Nei also proceeds through several chemical steps: N-glycosidic bond breakage, β-elimination and δ-elimination of the phosphates flanking the lesion. We have used stopped-flow kinetics with fluorescence detection to follow conformational changes in the Nei molecule when the enzyme binds normal DNA, damaged but uncleavable DNA, or several cleavable damaged DNA substrates. Binding normal or damaged uncleavable DNA proceeded in two fluorescently discernible reversible stages, while processing of cleavable substrates involved three reversible stages followed by and irreversible stage and equilibrium with the reaction product. Individual rate constants were calculated for each reaction step. Based on the stopped-flow data, crystal structure, and a comparison with the stopped-flow kinetics of E. coli formamidopyrimidine-DNA glycosylase, a homolog of Nei, we propose the nature of some of the steps that may be involved into the recognition and excision of damaged bases by Nei. [Copyright &y& Elsevier]