Search Results (24)

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. Full article

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. Full article

The β-adrenergic drug Mirabegron, a drug initially used for the treatment of an overactive bladder, has new potential indications and is hydrolyzed by butyrylcholinesterase (BChE). This compound is one of the only arylacylamide substrates to be catabolized by BChE. A steady-state kinetic analysis at 25 °C and pH 7.0 showed that the enzyme behavior is Michaelian with this substrate and displays a long pre-steady-state phase characterized by a burst. The induction time, τ, increased with substrate concentration (τ ≈ 18 min at maximum velocity). The kinetic behavior was interpreted in terms of hysteretic behavior, resulting from a slow equilibrium between two enzyme active forms, E and E′. The pre-steady-state phase with the highest activity corresponds to action of the E form, and the steady state corresponds to action of the E′ form. The catalytic parameters were determined as k_cat = 7.3 min⁻¹ and K_m = 23.5 μM for the initial (burst) form E, and k_cat = 1.6 min⁻¹ and K_m = 3.9 μM for the final form E′. Thus, the higher affinity of E′ for Mirabegron triggers the slow enzyme state equilibrium toward a slow steady state. Despite the complexity of the reaction mechanism of Mirabegron with BChE, slow BChE-catalyzed degradation of Mirabegron in blood should have no impact on the pharmacological activities of this drug. Full article

Base excision repair (BER), which involves the sequential activity of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases, is one of the enzymatic systems that preserve the integrity of the genome. Normal BER is effective, but due to single-nucleotide polymorphisms (SNPs), the enzymes themselves—whose main function is to identify and eliminate damaged bases—can undergo amino acid changes. One of the enzymes in BER is DNA polymerase β (Polβ), whose function is to fill gaps in DNA. SNPs can significantly affect the catalytic activity of an enzyme by causing an amino acid substitution. In this work, pre-steady-state kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring variants of Polβ that have the substitutions L19P and G66R in the dRP-lyase domain. Despite the substantial distance between the dRP-lyase domain and the nucleotidyltransferase active site, it was found that the capacity to form a complex with DNA and with an incoming dNTP is significantly altered by these substitutions. Therefore, the lower activity of the tested polymorphic variants may be associated with a greater number of unrepaired DNA lesions. Full article

This review deals with modern approaches to systematic research on molecular-kinetic mechanisms of damage recognition and removal by pro- and eukaryotic enzymes of DNA base excision repair. To this end, using DNA glycosylases from different structural families as an example—as well as apurinic/apyrimidinic endonuclease, which differs structurally and catalytically from DNA glycosylases—a comprehensive methodology is described in detail regarding studies on the mechanisms of action of DNA repair enzymes in humans and in Escherichia coli. This methodology is based on kinetic, thermodynamic, and mutational analyses of alterations in the conformation of molecules of an enzyme and of DNA during their interaction in real time. The described techniques can be used to analyze any protein–protein or protein–nucleic acid interactions. Full article

In the crystal structure of ferredoxin-NADP⁺ oxidoreductase from Bacillus subtilis (BsFNR), Tyr50 stacks on the si-face of the isoalloxazine ring portion of the FAD prosthetic group. This configuration is highly conserved among the homodimeric ferredoxin-NAD(P)⁺ oxidoreductases (FNR) from Gram-positive bacteria and photosynthetic bacteria. In this report, pre-steady state reactions of Tyr50 variants with NADP⁺/NADPH and ferredoxin from B. subtilis (BsFd) were examined with stopped-flow spectrophotometry to assess the effects of the mutation on the formation of FNR-substrate complexes and following redox equivalent transfer. Mixing oxidized BsFNRs with NADPH resulted in a rapid complex formation followed by a rate-limiting hydride transfer. The substitution substantially modulated the intensity of the charge transfer absorption band and decreased the observed hydride transfer rates compared to the wild type. Reduction of the Y50W mutant by NADPH proceeded in a monophasic manner, while the Y50G and Y50S mutants did in biphasic phases. The reduced Tyr50 mutants hardly promoted the reduction of NADP⁺. Mixing oxidized BsFNRs with reduced BsFd resulted in the reduction of the FNRs. The observed FNR reduction rates of the three variants were comparable, but in the Y50G and Y50S mutants, the amount of the reduced FNR at the rapid phase was decreased, and a slow FNR reduction phase was observed. The obtained results suggest that the replacements of Tyr50 with Gly and Ser permitted the conformational change in the reduced form, which induced an asymmetric kinetic behavior between the protomers of the homodimeric BsFNR. Full article

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. Full article

Human Fe(II)/α-ketoglutarate-dependent dioxygenase ABH2 plays a crucial role in the direct reversal repair of nonbulky alkyl lesions in DNA nucleobases, e.g., N¹-methyladenine (m¹A), N³-methylcytosine (m³C), and some etheno derivatives. Moreover, ABH2 is capable of a less efficient oxidation of an epigenetic DNA mark called 5-methylcytosine (m⁵C), which typically is a specific target of DNA dioxygenases from the TET family. In this study, to elucidate the mechanism of the substrate specificity of ABH2, we investigated the role of several active-site amino acid residues. Functional mapping of the lesion-binding pocket was performed through the analysis of the functions of Tyr122, Ile168, and Asp173 in the damaged base recognition mechanism. Interactions of wild-type ABH2, or its mutants Y122A, I168A, or D173A, with damaged DNA containing the methylated base m¹A or m³C or the epigenetic marker m⁵C were analyzed by molecular dynamics simulations and kinetic assays. Comparative analysis of the enzymes revealed an effect of the substitutions on DNA binding and on catalytic activity. Obtained data clearly demonstrate the effect of the tested amino acid residues on the catalytic activity of the enzymes rather than the DNA-binding ability. Taken together, these data shed light on the molecular and kinetic consequences of the substitution of active-site residues for the mechanism of the substrate recognition. Full article

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. Full article

To maintain the integrity of the genome, there is a set of enzymatic systems, one of which is base excision repair (BER), which includes sequential action of DNA glycosylases, apurinic/apyrimidinic endonucleases, DNA polymerases, and DNA ligases. Normally, BER works efficiently, but the enzymes themselves (whose primary function is the recognition and removal of damaged bases) are subject to amino acid substitutions owing to natural single-nucleotide polymorphisms (SNPs). One of the enzymes in BER is DNA polymerase β (Polβ), whose function is to fill gaps in DNA with complementary dNMPs. It is known that many SNPs can cause an amino acid substitution in this enzyme and a significant decrease in the enzymatic activity. In this study, the activity of four natural variants of Polβ, containing substitution E154A, G189D, M236T, or R254I in the transferase domain, was analyzed using molecular dynamics simulations and pre-steady-state kinetic analyses. It was shown that all tested substitutions lead to a significant reduction in the ability to form a complex with DNA and with incoming dNTP. The G189D substitution also diminished Polβ catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome. Full article

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an AP site, thereby forming a single–strand break containing 3′–OH and 5′–dRP ends. In addition, Apn2 has 3′–phosphodiesterase activity (removing 3′–blocking groups) and 3′ → 5′ exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3′–5′–exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single–strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped–flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5′ overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate–limiting step in the enzymatic reaction. We determined an influence of the nature of the 3′–terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context–specific. Full article

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. Full article

Elucidation of physicochemical mechanisms of enzymatic processes is one of the main tasks of modern biology. High efficiency and selectivity of enzymatic catalysis are mostly ensured by conformational dynamics of enzymes and substrates. Here, we applied a stopped-flow kinetic analysis based on fluorescent spectroscopy to investigate mechanisms of conformational transformations during the removal of alkylated bases from DNA by ALKBH2, a human homolog of Escherichia coli AlkB dioxygenase. This enzyme protects genomic DNA against various alkyl lesions through a sophisticated catalytic mechanism supported by a cofactor (Fe(II)), a cosubstrate (2-oxoglutarate), and O₂. We present here a comparative study of conformational dynamics in complexes of the ALKBH2 protein with double-stranded DNA substrates containing N1-methyladenine, N3-methylcytosine, or 1,N6-ethenoadenine. By means of fluorescent labels of different types, simultaneous detection of conformational transitions in the protein globule and DNA substrate molecule was performed. Fitting of the kinetic curves by a nonlinear-regression method yielded a molecular mechanism and rate constants of its individual steps. The results shed light on overall conformational dynamics of ALKBH2 and damaged DNA during the catalytic cycle. Full article

The Michaelis–Menten model of enzyme kinetic assumes the free ligand approximation, the steady-state approximation and the rapid equilibrium approximation. Analytical methods to model slow-binding inhibitors by the analysis of initial velocities have been developed but, due to their inherent complexity, they are seldom employed. In order to circumvent the complications that arise from the violation of the rapid equilibrium assumption, inhibition is commonly evaluated by pre-incubating the enzyme and the inhibitors so that, even for slow inhibitors, the binding equilibrium is established before the reaction is started. Here, we show that for long drug-target residence time inhibitors, the conventional analysis of initial velocities by the linear regression of double-reciprocal plots fails to provide a correct description of the inhibition mechanism. As a case study, the inhibition of acetylcholinesterase by galantamine, a drug approved for the symptomatic treatment of Alzheimer’s disease, is reported. For over 50 years, analysis based on the conventional steady-state model has overlooked the time-dependent nature of galantamine inhibition, leading to an erroneous assessment of the drug potency and, hence, to discrepancies between biochemical data and the pharmacological evidence. Re-examination of acetylcholinesterase inhibition by pre-steady state analysis of the reaction progress curves showed that the potency of galantamine has indeed been underestimated by a factor of ~100. Full article

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is ‘nicked’ rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I’s binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs. Full article