Antibiotic prescribing practices and perceptions on antimicrobial resistance among healthcare practitioners in Russia.

OBJECTIVES: Antimicrobial resistance (AMR) is a growing global health threat. The misuse of antibiotics is the main factor contributing to the development of AMR. Healthcare practitioners (HCPs) play a crucial role in the use of antibiotics. There are limited data available on antibiotic prescribing patterns among physicians in Russia. The aim of this study was to explore antibiotic prescribing practices and perceptions of AMR among HCPs in the Russian Federation. STUDY DESIGN: A cross-sectional, multi-centre study was used. METHODS: A survey using an online questionnaire was conducted among HCPs. A qualitative study was conducted before the questionnaire was developed. The online questionnaire was distributed via email addresses obtained from the Consilium Medicum database, a specialised educational resource for healthcare professionals in Russia. RESULTS: In total, 746 HCPs from 74 regions of the Russian Federation were included in the study. Physicians who participated in this study did not frequently prescribe antibiotic drugs: 40.6% of participants recommended antibiotics less than five times per week. Gynaecologists, paediatricians, family doctors, and surgeons were the least likely study participants to prescribe antibiotics, whereas clinical pharmacologists, otolaryngologists, urologists, and infectious disease specialists prescribed antibiotics more often. Amoxicillin and amoxicillin/clavulanic acid were the most frequently prescribed antimicrobials. The majority of HCPs in Russia who took part in this survey reported relying on national guidelines for information on antibiotic prescribing. Only 67.8% of study participants perceived AMR as a challenge for their practice. CONCLUSIONS: Health authorities should regularly provide up-to-date reliable information on AMR in the region. Antimicrobial stewardship programmes are important for specialised medical professionals, such as urologists, gynaecologists, and otolaryngologists, since they are responsible for prescribing second-line antibiotics, which carries with it a greater responsibility.

[The present-day epidemiology: challenges of public health and possibilities to settle them: the publications review].

Actually, the epidemiology is a dynamically developing medical science located at the intersection of social and biological branches of knowledge and bio-informatics. The new sources of data, the new methods create unique opportunities for epidemiologist. The number of epidemiological studies carrying out at the junction of several adjacent disciplines is increasing that requires harmonious interaction of specialists of different branches of medical knowledge. The change of the structure of global mortality towards chronic non-communicable diseases significantly affected the vector of epidemiological studies. Many interventional epidemiological projects are targeted to evaluation of effectiveness of new methods of prevention of cardiovascular, metabolic and oncological diseases. However, in recent years, the fight against unremembered infections affecting about 1 billion of people and taking away lives of 0.5 million people annually gained new importance. The current COVID-19 pandemic also affected epidemiology of communicable and chronic non-communicable diseases. Great attention is also currently attended to studying influence of social, economic and environmental factors on human health. The increase of average life expectancy of population contributes to development of epidemiology of the elderly. The new projects are initiated in the field of pharmacoepidemiology targeted to studying effectiveness of medications. The review of national and foreign publications considering current trends and achievements in the field of epidemiology. The reference retrieval engines such as PubMed, Google Scholar, CyberLeninka were used. The current directions of epidemiological research are analyzed. The challenges and development prospects of development of modern epidemiology are highlighted.

[Modern Approaches to Protein Engineering to Create Enzymes with New Catalytic Properties].

Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. Rational design was carried out to construct mutant enzyme forms with altered catalytic activity. Structures of the MutY mutants were calculated by molecular dynamics (MD). Their analysis showed that some of the MutY mutants may have AP lyase activity in addition to hydrolyzing the N-glycosidic bond, as is the case with bifunctional DNA glycosylases. MutY mutants with the A120K or S124K substitution were obtained by site-directed mutagenesis, and their catalytic activities were determined. The S120K substitution was shown to confer additional AP lyase activity, while the A124K substitution completely inactivated the enzyme.

[DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells].

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

[Medical and social assessment of the quality of life of a family with a child suffering from food allergy].

Food allergy (FA) is a health problem that adversely affect the quality of life of children and their family members. The purpose of the study was to assess the quality of life in families with children affected FA. Material and methods. A cross-sectional study was conducted in a group of 75 children with a confirmed FA (at the age of Me 4.9 years [1.3; 7.1]). One of the caregivers of the child was asked to complete the Russian version of a specialized questionnaire «The Food hypersensitivity famiLy ImPact, FLIP¼ for assessing the life quality of families with children affected FA. Results. Diet organization is the main concern affecting quality of life, while the daily life of the family and the emotional sphere are less impacted. Age, type of food allergens and clinical manifestations do not significantly contribute the life quality indicators. Hypersensitivity to several food is statistically associated with changes in everyday life and emotions. Non-compliance with the diet is associated with a lower impact of FA on quality of life. 56% of respondents worried about the nutritional value of child's diet and 49.3% of caregivers reported that a child's FA significantly impacted grocery shopping behaviors (reading labels, etc.). At the same time, 73.3% noted that child's FA does not affect the diet of other family members. Also, 33.3% of the parents experienced anxiety due to child's FA and 38.7% are worried that FA might stay persistent. 30.7% of respondents are afraid of accidental consuming of allergenic products. Conclusion. The acquired results indicate the importance of quality of life assessment for understanding the social aspects of FA. Strategies to improve the quality of life include the development of informational and educational programs both for parents and patients. In order to estimate impact of FA to life quality from the patient's perspective further development of questionnaires adapted for children and adolescents is necessary.

[Methods of gut microbiota correction for treatment and prevention of food allergy: a review of current research].

Food allergy (FA) is an actual problem in pediatric practice. The gut microbiota plays a crucial role in food sensitization development, since the maturation of immune system occurs under the influence of intestinal microorganisms. Immunoregulatory activity of gut microbiota is associated with the increase of IgA production and promotion of the barrier function of intestinal epithelium. Gut microbiota influence the activity of T-regulatory cells, as well. Violation of gut biocenosis, which occurs under the influence of various factors (artificial feeding, past diseases, the use of antibiotics, etc.), can lead to a shift in the balance of the immune system towards the increase of Th2-profile cytokines and the subsequent formation of hypersensitivity to food allergens. In this regard, the correction of the gut microbiome is a promising method of FA control, due to the ability of intestinal bacteria influence the production of T-regulatory cells and thus suppress allergy immune response. The aim of the review is to analyze experimental and clinical studies exploring effectiveness of methods modifying intestinal microbiota in order to treat and prevent FA. Material and methods. The analysis of the literature in eLIBRARY, MedLine and PubMed databases was carried out. Results. The analysis revealed the lack of rigorous evidence that pre-, pro- and synbiotics significantly increase the effectiveness of standard therapy of FA. However, the use of bifidobacteria, lactobacilli, lactic acid bacteria, in combination with the basic therapy of FA has general positive effect on the clinical outcome, especially in case of gastrointestinal symptoms. Also, the results of some studies indicate the effectiveness of synbiotics (Bifidobacterium breve M-16V, Lactobacillus rhamnosus GG in combination with oligosaccharides) for the prevention of FA in patients at risk of developing allergic diseases in the long-term period. Conclusion. At present, fecal microbiota transplantation is promising method for FA treatment. Polysaccharides fermented by the microflora, are also actively studied. Experimental studies and clinical trials are required to obtain substantiated conclusions about feasibility of these methods for treatment and prevention of FA.

Trends in Population-Based Studies: Molecular and Digital Epidemiology (Review).

The development of high-throughput technologies has sharply increased the opportunities to research the human body at the molecular, cellular, and organismal levels in the last decade. Rapid progress in biotechnology has caused a paradigm shift in population-based studies. Advances in modern biomedical sciences, including genomic, genome-wide, post-genomic research and bioinformatics, have contributed to the emergence of molecular epidemiology focused on the study of the personalized molecular mechanism of disease development and its extrapolation to the population level. The work of research teams at the intersection of information technology and medicine has become the basis for highlighting digital epidemiology, the important tools of which are machine learning, the ability to work with real world data, and accumulated big data. The developed approaches accelerate the process of collecting and processing biomedical data, testing new scientific hypotheses. However, new methods are still in their infancy, they require testing of application under various conditions, as well as standardization. This review highlights the role of omics and digital technologies in population-based studies.

Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity.

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg2+-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg2+ ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

[Initial stages of DNA Base Excision Repair in Nucleosomes].

In mammalian cells, base excision repair (BER) is the main pathway responsible for the correction of a variety of chemically modified DNA bases. DNA packaging in chromatin affects the accessibility of damaged sites to the enzymes involved in repair processes. This review presents data concerning the enzymes involved in BER. Within the nucleosome core particle (NCP), the accessibility of damaged DNA to enzymes is hindered by the presence of a histone octamer. This means that the removal of DNA lesions largely depends on their rotational and translational positioning in the NCP, as well as on the specific features of each enzyme.

[Mutational and Kinetic Analysis of APE1 Endoribonuclease Activity].

Human apurinic/apyrimidinic endonuclease 1 (APE1) participates in the DNA repair system. It is believed that the main biological function of APE1 is Mg^(2+)-dependent hydrolysis of AP-sites in DNA. On the base of structural data, kinetic studies, and mutation analysis, the key stages of APE1 interaction with damaged DNA were established. It has been shown recently that APE1 can act as an endoribonuclease that catalyzes mRNA hydrolysis at certain pyrimidine-purine sites and thus controls the level of certain transcripts. In addition, the presence of Mg^(2+) ions was shown to be not required for the endoribonuclease activity of APE1, in contrast to the AP-endonuclease activity. This indicates differences in mechanisms of APE1 catalysis on RNA and DNA substrates, but the reasons for these differences remain unclear. Here, the analysis of endoribonuclease hydrolysis of model RNA substrates with wild type APE1 enzyme and its mutant forms Y171F, R177F, R181A, D210N, N212A, T268D, M270A, and D308A, was performed. It was shown that mutation of Asn212, Asp210, and Tyr171 residues leads to the decrease of AP-endonuclease activity while endoribonuclease activity is retained. Also, T268D and M270A APE1 mutants lose specificity to pyrimidine-purine sequences. R177F and R181A did not show a significant decrease in enzyme activity, whereas D308A demonstrated a decrease of endoribonuclease activity.

[Comparative Analysis of the Activity of the Polymorphic Variants of Human Uracil-DNA-Glycosylases SMUG1 and MBD4].

The human N-glycosylases SMUG1 and MBD4 catalyze the removal of uracil residues from DNA resulting from cytosine deamination or replication errors. For polymorphic variants of SMUG1 (G90C, P240H, N244S, N248Y) and the MBD4^(cat) catalytic domain (S470L, G507S, R512W, H557D), the structures of enzyme-substrate complexes were obtained by molecular dynamic simulation. It was experimentally found that the SNP variants of SMUG1, N244S and N248Y, had increased catalytic activity compared to the wild-type enzyme, probably due to the acceleration of the dissociation of the enzyme-product complex and an increase in the enzyme turnover rate. All other SNP variants of SMUG1 (G90C, P240H) and MBD4^(cat), in which amino acid substitutions disrupted the substrate binding region and/or active site, had significantly lower catalytic activity than the wild-type enzymes.

[Efficiency of RNA Hydrolysis by Binase from Bacillus pumilus: The Impact of Substrate Structure, Metal Ions, and Low Molecular Weight Nucleotide Compounds].

Binase is an extracellular guanyl-preferring ribonuclease from Bacillus pumilus. The main biological function of binase is RNA degradation with the formation of guanosine-2',3'-cyclic phosphate and its subsequent hydrolysis to 3'-phosphate. Extracellular RNases are believed to be key agents that affect the functional activity of the body, as they directly interact with epithelial and immune cells. The biological effects of the enzyme may consist of both direct RNA degradation, and the accumulation of 2',3'-cGMP in the human body. In this work, we have performed a comparative analysis of the cleavage efficiency of model RNA substrates, i.e., short hairpin structures that contain guanosine at various positions. It has been shown that the hydrolysis efficiency of the model RNA substrates depends on the position of guanosine. We have also demonstrated the influence of various divalent metal ions and low molecular weight nucleotide compounds on the binase-catalyzed endoribonucleolytic reaction.

The role of active-site amino acid residues in the cleavage of DNA and RNA substrates by human apurinic/apyrimidinic endonuclease APE1.

BACKGROUND: Human apurinic/apyrimidinic endonuclease APE1 is one of participants of the DNA base excision repair pathway. APE1 processes AP-sites and many other types of DNA damage via hydrolysis of the phosphodiester bond on the 5' side of the lesion. APE1 also acts as an endoribonuclease, i.e., can cleave undamaged RNA. METHODS: Using pre-steady-state kinetic analysis we examined the role of certain catalytically important amino acids in APE1 enzymatic pathway and described their involvement in the mechanism of the target nucleotide recognition. RESULTS: Comparative analysis of the cleavage efficiency of damaged DNAs containing an abasic site, 5,6-dihydrouridine, or α-anomer of adenosine as well as 3'-5'-exonuclease degradation of undamaged DNA and endonuclease hydrolysis of RNA substrates by mutant APE1 enzymes containing a substitution of an active-site amino acid residue (D210N, N212A, T268D, M270A, or D308A) was performed. Detailed pre-steady-state kinetics of conformational changes of the enzyme and of DNA substrate molecules during recognition and cleavage of the abasic site were studied. CONCLUSIONS: It was revealed that substitution T268D significantly disturbed initial DNA binding, whereas Asn212 is critical for the DNA-bending stage and catalysis. Substitution D210N increased the binding efficacy and blocked the catalytic reaction, but D308A decreased the binding efficacy owing to disruption of Mg2+ coordination. Finally, the substitution of Met270 also destabilized the enzyme-substrate complex but did not affect the catalytic reaction. SIGNIFICANCE: It was found that the tested substitutions of the active-site amino acid residues affected different stages of the complex formation process as well as the catalytic reaction.

Effect of the Substrate Structure and Metal Ions on the Hydrolysis of Undamaged RNA by Human AP Endonuclease APE1.

Human apurinic/apyrimidinic (AP) endonuclease APE1 is one of the participants in the DNA base excision repair. The main biological function of APE1 is to hydrolyze the phosphodiester bond on the 5'-side of the AP sites. It has been shown recently that APE1 acts as an endoribonuclease and can cleave mRNA, thereby controlling the level of some transcripts. The sequences of CA, UA, and UG dinucleotides are the cleavage sites in RNA. In the present work, we performed a comparative analysis of the cleavage efficiency of model RNA substrates with short hairpin structures in which the loop size and the location of the pyrimidine-purine dinucleotide sequence were varied. The effect of various divalent metal ions and pH on the efficiency of the endoribonuclease reaction was analyzed. It was shown that site-specific hydrolysis of model RNA substrates depends on the spatial structure of the substrate. In addition, RNA cleavage occured in the absence of divalent metal ions, which proves that hydrolysis of DNA- and RNA substrates occurs via different catalytic mechanisms.

Role of Arg243 and His239 Residues in the Recognition of Damaged Nucleotides by Human Uracil-DNA Glycosylase SMUG1.

Human uracil-DNA glycosylase SMUG1 removes uracil residues and some other noncanonical or damaged bases from DNA. Despite the functional importance of this enzyme, its X-ray structure is still unavailable. Previously, we performed homology modeling of human SMUG1 structure and suggested the roles of some amino acid residues in the recognition of damaged nucleotides and their removal from DNA. In this study, we investigated the kinetics of conformational transitions in the protein and in various DNA substrates during enzymatic catalysis using the stopped-flow method based on changes in the fluorescence intensity of enzyme's tryptophan residues and 2-aminopurine in DNA or fluorescence resonance energy transfer (FRET) between fluorophores in DNA. The kinetic mechanism of interactions between reaction intermediates was identified, and kinetic parameters of the intermediate formation and dissociation were calculated. The obtained data help in elucidating the functions of His239 and Arg243 residues in the recognition and removal of damaged nucleotides by SMUG1.

An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes.

Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5'-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

Thermodynamics of the DNA Repair Process by Endonuclease VIII.

In the present work, a thermodynamic analysis of the interaction between endonuclease VIII (Endo VIII) and model DNA substrates containing damaged nucleotides, such as 5,6-dihydrouridine and 2-hydroxymethyl-3-hydroxytetrahydrofuran (F-site), was performed. The changes in the fluorescence intensity of the 1,3-diaza-2-oxophenoxazine (tC°) residue located in the complementary chain opposite to the specific site were recorded in the course of the enzyme-substrate interaction. The kinetics was analyzed by the stopped-flow method at different temperatures. The changes of standard Gibbs free energy, enthalpy, and entropy of sequential steps of DNA substrate binding, as well as activation enthalpy and entropy for the transition complex formation of the catalytic stage, were calculated. The comparison of the kinetic and thermodynamic data characterizing the conformational transitions of enzyme and DNA in the course of their interaction made it possible to specify the nature of the molecular processes occurring at the stages of substrate binding, recognition of the damaged base, and its removal from DNA.

Global DNA dynamics of 8-oxoguanine repair by human OGG1 revealed by stopped-flow kinetics and molecular dynamics simulation.

The toxic action of different endogenous and exogenous agents leads to damage in genomic DNA. 8-Oxoguanine is one of the most often generated and highly mutagenic oxidative forms of damage in DNA. Normally, in human cells it is promptly removed by 8-oxoguanine-DNA-glycosylase hOGG1, the key DNA-repair enzyme. An association between the accumulation of oxidized guanine and an increased risk of harmful processes in organisms was already found. However, the detailed mechanism of damaged base recognition and removal is still unclear. To clarify the role of active site amino acids in the damaged base coordination and to reveal the elementary steps in the overall enzymatic process we investigated hOGG1 mutant forms with substituted amino acid residues in the enzyme base-binding pocket. Replacing the functional groups of the enzyme active site allowed us to change the rates of the individual steps of the enzymatic reaction. To gain further insight into the mechanism of hOGG1 catalysis a detailed pre-steady state kinetic study of this enzymatic process was carried out using the stopped-flow approach. The changes in the DNA structure after mixing with enzymes were followed by recording the FRET signal using Cy3/Cy5 labels in DNA substrates in the time range from milliseconds to hundreds of seconds. DNA duplexes containing non-damaged DNA, 8-oxoG, or an AP-site or its unreactive synthetic analogue were used as DNA-substrates. The kinetic parameters of DNA binding and damage processing were obtained for the mutant forms and for WT hOGG1. The analyses of fluorescence traces provided information about the DNA dynamics during damage recognition and removal. The kinetic study for the mutant forms revealed that all introduced substitutions reduced the efficiency of the hOGG1 activity; however, they played pivotal roles at certain elementary stages identified during the study. Taken together, our results gave the opportunity to restore the role of substituted amino acids and main "damaged base-amino acid" contacts, which provide an important link in the understanding the mechanism of the DNA repair process catalyzed by hOGG1.

Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4.

The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme's amino acids.

The formation of catalytically competent enzyme-substrate complex is not a bottleneck in lesion excision by human alkyladenine DNA glycosylase.

Human alkyladenine DNA glycosylase (AAG) protects DNA from alkylated and deaminated purine lesions. AAG flips out the damaged nucleotide from the double helix of DNA and catalyzes the hydrolysis of the N-glycosidic bond to release the damaged base. To understand better, how the step of nucleotide eversion influences the overall catalytic process, we performed a pre-steady-state kinetic analysis of AAG interaction with specific DNA-substrates, 13-base pair duplexes containing in the 7th position 1-N6-ethenoadenine (εA), hypoxanthine (Hx), and the stable product analogue tetrahydrofuran (F). The combination of the fluorescence of tryptophan, 2-aminopurine, and 1-N6-ethenoadenine was used to record conformational changes of the enzyme and DNA during the processes of DNA lesion recognition, damaged base eversion, excision of the N-glycosidic bond, and product release. The thermal stability of the duplexes characterized by the temperature of melting, Tm, and the rates of spontaneous opening of individual nucleotide base pairs were determined by NMR spectroscopy. The data show that the relative thermal stability of duplexes containing a particular base pair in position 7, (Tm(F/T) < Tm(εA/T) < Tm(Hx/T) < Tm(A/T)) correlates with the rate of reversible spontaneous opening of the base pair. However, in contrast to that, the catalytic lesion excision rate is two orders of magnitude higher for Hx-containing substrates than for substrates containing εA, proving that catalytic activity is not correlated with the stability of the damaged base pair. Our study reveals that the formation of the catalytically competent enzyme-substrate complex is not the bottleneck controlling the catalytic activity of AAG.