Opisthorchis Felineus Infection is a Risk Factor for Cholangiocarcinoma in Western Siberia: A Hospital-based Case-control Study.

BACKGROUND: Cholangiocarcinoma (CCA), a fatal bile duct cancer, has a high incidence in Western Siberia, Russian Federation. In addition, Opisthorchis felineus, a bile duct-dwelling trematode liver fluke is highly endemic. Closely related species have been shown to be cancerogenic agents in Asia. We therefore examined the association between O felineus infection and CCA in Western Siberia. METHODS: We conducted a hospital-based, individually matched case-control study between January 2017 and August 2020 in Tomsk Oblast and Khanty-Mansiysk Autonomous Okrug, Yugra, Russian Federation. Histologically confirmed CCA patients (cases) were compared with matched age, sex, and place of residence hospital controls. The examination of study participants included the diagnosis of current and past O felineus infection, abdominal ultrasonographical assessment, physical examination, and interview on exposures to potential risk factors. RESULTS: We identified 40 patients with CCA and 160 controls. Exposures to O felineus infection was strongly associated with CCA (odds ratio [OR], 3.9; 95% confidence interval [CI], 1.4-10.8; P = .008). Also, cases reported more often that they were currently or in the past were infected by O felineus compared with controls (OR, 4.03; 95% CI, 1.7-9.5; P = .001). Furthermore, cases reported river fish consumption and fishing habits significantly more often than controls (OR, 5.5; 95% CI, 1.5-19.8; P = .009 and OR, 3.3; 95% CI, 1.4-7.7; P = .005). CONCLUSIONS: The study results revealed a strong significantly increased risk for CCA development in O felineus-infected individuals. Elaboration of the guidelines on screening programs for early CCA diagnosis, prevention, and treatment is socially important in endemic regions.

One-Pot Radiosynthesis of [18F]Anle138b-5-(3-Bromophenyl)-3-(6-[18F]fluorobenzo[d][1,3]dioxol-5-yl)-1H-pyrazole-A Potential PET Radiotracer Targeting α-Synuclein Aggregates.

Availability of PET imaging radiotracers targeting α-synuclein aggregates is important for early diagnosis of Parkinson's disease and related α-synucleinopathies, as well as for the development of new therapeutics. Derived from a pyrazole backbone, 11C-labelled derivatives of anle138b (3-(1,3-benzodioxol-5-yl)-5-(3-bromophenyl)-1H-pyrazole)-an inhibitor of α-synuclein and prion protein oligomerization-are currently in active development as the candidates for PET imaging α-syn aggregates. This work outlines the synthesis of a radiotracer based on the original structure of anle138b, labelled with fluorine-18 isotope, eminently suitable for PET imaging due to half-life and decay energy characteristics (97% ß+ decay, 109.7 min half-life, and 635 keV positron energy). A three-step radiosynthesis was developed starting from 6-[18F]fluoropiperonal (6-[18F]FP) that was prepared using (piperonyl)(phenyl)iodonium bromide as a labelling precursor. The obtained 6-[18F]FP was used directly in the condensation reaction with tosylhydrazide followed by 1,3-cycloaddition of the intermediate with 3'-bromophenylacetylene eliminating any midway without any intermediate purifications. This one-pot approach allowed the complete synthesis of [18F]anle138b within 105 min with RCY of 15 ± 3% (n = 3) and Am in the range of 32-78 GBq/µmol. The [18F]fluoride processing and synthesis were performed in a custom-built semi-automated module, but the method can be implemented in all the modern automated platforms. While there is definitely space for further optimization, the procedure developed is well suited for preclinical studies of this novel radiotracer in animal models and/or cell cultures.

Pharmacokinetics of Ascending Doses of Praziquantel in Adults Infected with Opisthorchis felineus in Western Siberia, Russian Federation.

Opisthorchiasis due to the liver fluke Opisthorchis felineus is highly prevalent in rural regions of Western Siberia, causing severe liver and bile duct maladies. Praziquantel administered as a three-dose regimen is the only drug used to treat O. felineus-infected individuals. A simpler single-dose treatment might serve as an alternative. The aim of this study was to compare the pharmacokinetic (PK) properties of single, ascending doses of praziquantel compared to multiple dosing in patients infected with O. felineus to contribute to updated treatment guidelines. Dried blood spots (DBSs) of 110 adults were collected at 11 time points post-drug administration at single oral doses of 20, 40, and 60 mg/kg, as well as 3× 20 mg/kg (4 h dosing interval). DBS samples were analyzed using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, and PK parameters were obtained for R-, S-, and R-trans-4-OH-praziquantel employing noncompartmental analysis. We observed the highest drug exposure for all analytes when the triple-dose scheme was used; area under the concentration-time curve from 0 to 24 h (AUC0-24) values of 8.04, 27.75, and 36.38 µg/mL·h were obtained, respectively. Maximal plasma concentrations (Cmax) values of 1.72, 4.89, and 2.69 µg/mL were calculated for R-, S-, and R-trans-4-OH-praziquantel, respectively, when patients were given a single 60-mg/kg dose, and they peaked at 1.5 and 2 h for the enantiomers and at 3 h for the metabolite. The herein-generated PK data, together with results that will be obtained from the integrated efficacy study, lay the groundwork for a possibly optimized treatment scheme for O. felineus-infected patients.

Insights into Mechanisms of Damage Recognition and Catalysis by APE1-like Enzymes.

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.

Structural and Molecular Kinetic Features of Activities of DNA Polymerases.

DNA polymerases catalyze DNA synthesis during the replication, repair, and recombination of DNA. Based on phylogenetic analysis and primary protein sequences, DNA polymerases have been categorized into seven families: A, B, C, D, X, Y, and RT. This review presents generalized data on the catalytic mechanism of action of DNA polymerases. The structural features of different DNA polymerase families are described in detail. The discussion highlights the kinetics and conformational dynamics of DNA polymerases from all known polymerase families during DNA synthesis.

The Role of Natural Polymorphic Variants of DNA Polymerase ß in DNA Repair.

DNA polymerase ß (Polß) is considered the main repair DNA polymerase involved in the base excision repair (BER) pathway, which plays an important part in the repair of damaged DNA bases usually resulting from alkylation or oxidation. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that protein-protein interactions of Polß with enzymes from the BER pathway increase the efficiency of damaged base repair in DNA. However natural single-nucleotide polymorphisms can lead to a substitution of functionally significant amino acid residues and therefore affect the catalytic activity of the enzyme and the accuracy of Polß action. Up-to-date databases contain information about more than 8000 SNPs in the gene of Polß. This review summarizes data on the in silico prediction of the effects of Polß SNPs on DNA repair efficacy; available data on cancers associated with SNPs of Polß; and experimentally tested variants of Polß. Analysis of the literature indicates that amino acid substitutions could be important for the maintenance of the native structure of Polß and contacts with DNA; others affect the catalytic activity of the enzyme or play a part in the precise and correct attachment of the required nucleotide triphosphate. Moreover, the amino acid substitutions in Polß can disturb interactions with enzymes involved in BER, while the enzymatic activity of the polymorphic variant may not differ significantly from that of the wild-type enzyme. Therefore, investigation regarding the effect of Polß natural variants occurring in the human population on enzymatic activity and protein-protein interactions is an urgent scientific task.

Kinetic Features of 3'-5'-Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae.

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.

Comparative Analysis of Exo- and Endonuclease Activities of APE1-like Enzymes.

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.

Automated Optimized Synthesis of [18F]FLT Using Non-Basic Phase-Transfer Catalyst with Reduced Precursor Amount.

3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) is a positron emission tomography (PET) tracer useful for tumor proliferation assessment for a number of cancers, particularly in the cases of brain, lung, and breast tumors. At present [18F], FLT is commonly prepared by means of the nucleophilic radiofluorination of 3-N-Boc-5'-O-DMT-3'-O-nosyl thymidine precursor in the presence of a phase-transfer catalyst, followed by an acidic hydrolysis. To achieve high radiochemical yield, relatively large amounts of precursor (20−40 mg) are commonly used, leading to difficulties during purification steps, especially if a solid-phase extraction (SPE) approach is attempted. The present study describes an efficient method for [18F]FLT synthesis, employing tetrabutyl ammonium tosylate as a non-basic phase-transfer catalyst, with a greatly reduced amount of precursor employed. With a reduction of the precursor amount contributing to lower amounts of synthesis by-products in the reaction mixture, an SPE purification procedure using only two commercially available cartridges­OASIS HLB 6cc and Sep-Pak Alumina N Plus Light­has been developed for use on the GE TRACERlab FX N Pro synthesis module. [18F]FLT was obtained in radiochemical yield of 16 ± 2% (decay-corrected) and radiochemical purity >99% with synthesis time not exceeding 55 min. The product was formulated in 16 mL of normal saline with 5% ethanol (v/v). The amounts of chemical impurities and residual solvents were within the limits established by European Pharmacopoeia. The procedure described compares favorably with previously reported methods due to simplified automation, cheaper and more accessible consumables, and a significant reduction in the consumption of an expensive precursor.

Conformational Dynamics of Human ALKBH2 Dioxygenase in the Course of DNA Repair as Revealed by Stopped-Flow Fluorescence Spectroscopy.

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.

Obligatory medical prescription of antibiotics in Russia: Navigating formal and informal health-care infrastructures.

Antimicrobial resistance control programmes often aim to "fix" the behaviour of antibiotic users and prescribers. Such behavioural interventions have been widely criticised in social science literature for being inefficient and too narrow in scope. Drawing on these criticisms, this article analyses how political programmes for fixing antibiotic behaviours were adapted into the practices of health-care professionals and patients in Russia. In 2018, we conducted interviews with medical doctors, pharmacists and patients in a Russian city; focusing on their practices around the policy requirement introduced in 2017 which obligated medical prescriptions of antibiotics. We conceptualised the obligatory medical prescription as a political technique which sought to change practices of self-treatment and over-the-counter sales of medications by establishing doctors as an obligatory passage point to access antibiotics. Our analysis shows that the requirement for medical prescriptions does not fulfil the infrastructural gaps that influence antibiotic practices. By navigating the antibiotic prescriptions, doctors, pharmacists and patients informally compensate for the gaps in the existing infrastructure creating informal networks of antibiotic care parallel to the requirement of obligatory prescriptions. Following these informal practices, we could map the inconsistencies in the current policy approaches to tackle AMR as a behavioural rather than infrastructural problem.

DNA Demethylation in the Processes of Repair and Epigenetic Regulation Performed by 2-Ketoglutarate-Dependent DNA Dioxygenases.

Site-specific DNA methylation plays an important role in epigenetic regulation of gene expression. Chemical methylation of DNA, including the formation of various methylated nitrogenous bases, leads to the formation of genotoxic modifications that impair DNA functions. Despite the fact that different pathways give rise to methyl groups in DNA, the main pathway for their removal is oxidative demethylation, which is catalyzed by nonheme Fe(II)/α-ketoglutarate-dependent DNA dioxygenases. DNA dioxygenases share a common catalytic mechanism of the oxidation of the alkyl groups on nitrogenous bases in nucleic acids. This review presents generalized data on the catalytic mechanism of action of DNA dioxygenases and on the participation of typical representatives of this superfamily, such as prokaryotic enzyme AlkB and eukaryotic enzymes ALKBH1-8 and TET1-3, in both processes of direct repair of alkylated DNA adducts and in the removal of an epigenetic mark (5-methylcytosine).

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.

Production of 6-l-[18F]Fluoro-m-tyrosine in an Automated Synthesis Module for 11C-Labeling.

6-l-[18F]Fluoro-m-tyrosine (6-l-[18F]FMT) represents a valuable alternative to 6-l-[18F]FDOPA which is conventionally used for the diagnosis and staging of Parkinson's disease. However, clinical applications of 6-l-[18F]FMT have been limited by the paucity of practical production methods for its automated production. Herein we describe the practical preparation of 6-l-[18F]FMT using alcohol-enhanced Cu-mediated radiofluorination of Bpin-substituted chiral Ni(II) complex in the presence of non-basic Bu4ONTf using a volatile iPrOH/MeCN mixture as reaction solvent. A simple and fast radiolabeling procedure afforded the tracer in 20.0 ± 3.0% activity yield within 70 min. The developed method was directly implemented onto a modified TracerLab FX C Pro platform originally designed for 11C-labeling. This method enables an uncomplicated switch between 11C- and 18F-labeling. The simplicity of the developed procedure enables its easy adaptation to other commercially available remote-controlled synthesis units and paves the way for a widespread application of 6-l-[18F]FMT in the clinic.

Substrate specificity of human apurinic/apyrimidinic endonuclease APE1 in the nucleotide incision repair pathway.

Human apurinic/apyrimidinic (AP) endonuclease APE1 catalyses the hydrolysis of phosphodiester bonds on the 5' side of an AP-site (in the base excision repair pathway) and of some damaged nucleotides (in the nucleotide incision repair pathway). The range of substrate specificity includes structurally unrelated damaged nucleotides. Here, to examine the mechanism of broad substrate specificity of APE1, we performed pulsed electron-electron double resonance (PELDOR) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer (FRET) detection of DNA conformational changes during DNA binding and lesion recognition. Equilibrium PELDOR and kinetic FRET data revealed that DNA binding by APE1 leads to noticeable damage-dependent bending of a DNA duplex. Molecular dynamics simulations showed that the damaged nucleotide is everted from the DNA helix and placed into the enzyme's binding pocket, which is formed by Asn-174, Asn-212, Asn-229, Ala-230, Phe-266 and Trp-280. Nevertheless, no damage-specific contacts were detected between these amino acid residues in the active site of the enzyme and model damaged substrates containing 1,N6-ethenoadenosine, α-adenosine, 5,6-dihydrouridine or F-site. These data suggest that the substrate specificity of APE1 is controlled by the ability of a damaged nucleotide to flip out from the DNA duplex in response to an enzyme-induced DNA distortion.

Kinetic Milestones of Damage Recognition by DNA Glycosylases of the Helix-Hairpin-Helix Structural Superfamily.

X-ray data show that DNA glycosylases, which initiate the pathway of base excision repair in DNA, belong to six structural superfamilies. Here, we provide an overview of the latest results of kinetic studies on the mechanisms of specific recognition of a damaged nucleotide at the early steps of DNA repair by human (OGG1 and MBD4) or Escherichia coli (Nth and MutY) N-DNA-glycosylases belonging to superfamily Helix-hairpin-Helix (HhH). A comparison of real-time conformational transformations of DNA glycosylases and DNA with the structural data obtained for free enzymes and their complexes with substrates and intermediates have made it possible to build molecular-kinetic models of the enzymatic processes. These models have allowed researchers to associate the conformational transitions of the interacting molecules with elementary steps of an enzymatic process. Additionally, these models have revealed the stages that make the largest contribution to the specificity of the enzyme for DNA substrates. These data provide an opportunity to gain further insight into the structural and dynamic principles underlying the enzymatic processes that ensure highly efficient functioning of the repair-protective system of all living organisms and that maintain DNA integrity.

Modulation of the Apurinic/Apyrimidinic Endonuclease Activity of Human APE1 and of Its Natural Polymorphic Variants by Base Excision Repair Proteins.

Human apurinic/apyrimidinic endonuclease 1 (APE1) is known to be a critical player of the base excision repair (BER) pathway. In general, BER involves consecutive actions of DNA glycosylases, AP endonucleases, DNA polymerases, and DNA ligases. It is known that these proteins interact with APE1 either at upstream or downstream steps of BER. Therefore, we may propose that even a minor disturbance of protein-protein interactions on the DNA template reduces coordination and repair efficiency. Here, the ability of various human DNA repair enzymes (such as DNA glycosylases OGG1, UNG2, and AAG; DNA polymerase Polß; or accessory proteins XRCC1 and PCNA) to influence the activity of wild-type (WT) APE1 and its seven natural polymorphic variants (R221C, N222H, R237A, G241R, M270T, R274Q, and P311S) was tested. Förster resonance energy transfer-based kinetic analysis of abasic site cleavage in a model DNA substrate was conducted to detect the effects of interacting proteins on the activity of WT APE1 and its single-nucleotide polymorphism (SNP) variants. The results revealed that WT APE1 activity was stimulated by almost all tested DNA repair proteins. For the SNP variants, the matters were more complicated. Analysis of two SNP variants, R237A and G241R, suggested that a positive charge in this area of the APE1 surface impairs the protein-protein interactions. In contrast, variant R221C (where the affected residue is located near the DNA-binding site) showed permanently lower activation relative to WT APE1, whereas neighboring SNP N222H did not cause a noticeable difference as compared to WT APE1. Buried substitution P311S had an inconsistent effect, whereas each substitution at the DNA-binding site, M270T and R274Q, resulted in the lowest stimulation by BER proteins. Protein-protein molecular docking was performed between repair proteins to identify amino acid residues involved in their interactions. The data uncovered differences in the effects of BER proteins on APE1, indicating an important role of protein-protein interactions in the coordination of the repair pathway.

The Role of Active-Site Plasticity in Damaged-Nucleotide Recognition by Human Apurinic/Apyrimidinic Endonuclease APE1.

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.

A Single-Turnover Kinetic Study of DNA Demethylation Catalyzed by Fe(II)/α-Ketoglutarate-Dependent Dioxygenase AlkB.

AlkB is a Fe(II)/α-ketoglutarate-dependent dioxygenase that repairs some alkylated bases of DNA and RNA in Escherichia coli. In the course of catalysis, oxidation of a co-substrate (α-ketoglutarate, αKG) leads to the formation of a highly reactive 'oxyferryl' enzyme-bound intermediate, Fe(IV) = O, ensuring hydroxylation of the alkyl nucleobase adducts. Previous studies have revealed that AlkB is a flexible protein and can adopt different conformations during interactions with cofactors and DNA. To assess the conformational dynamics of the enzyme in complex with single- or double-stranded DNA in real-time mode, we employed the stopped-flow fluorescence method. N1-Methyladenine (m1A) introduced into a sequence of 15-mer oligonucleotides was chosen as the specific damage. Single-turnover kinetics were monitored by means of intrinsic fluorescence of the protein's Trp residues, fluorescent base analogue 2-aminopurine (2aPu), and a dye-quencher pair (FAM/BHQ1). For all the fluorescent labels, the fluorescent traces showed several phases of consistent conformational changes, which were assigned to specific steps of the enzymatic process. These data offer an overall picture of the structural dynamics of AlkB and DNA during their interaction.

The Role of Active-Site Residues Phe98, His239, and Arg243 in DNA Binding and in the Catalysis of Human Uracil-DNA Glycosylase SMUG1.

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme-DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme-DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.