[Comparative Analysis of Family A DNA-Polymerases as a Searching Tool for Enzymes with New Properties].

DNA polymerases catalyze DNA synthesis during DNA replication, repair, and recombination. A number of DNA polymerases, such as the Taq enzyme from Thermus aquaticus, are used in various applications of molecular biology and biotechnology, in particular as DNA amplification tools. However, the efficiency of these enzymes depends on factors such as DNA origin, primer composition, template length, GC-content, and the ability to form stable secondary structures. These limitations in the use of currently known DNA polymerases lead to the search for new enzymes with improved properties. This review summarizes the main structural and molecular-kinetic features of the functioning of DNA-polymerases belonging to structural family A, including Taq polymerase. A phylogenetic analysis of these enzymes was carried out, which made it possible to establish a highly conserved consensus sequence containing 62 amino acid residues distributed over the structure of the enzyme. A comparative analysis of these amino acid residues among poorly studied DNA-polymerases revealed 7 enzymes that potentially have the properties necessary for use in DNA amplification.

[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.

[Priorities of national surgery. Evgeny Illarionovich Zakharov. Stomach repair with small bowel substitutes]. / Prioritety otechestvennoi khirurgii. Evgenii Illarionovich Zakharov. Tonkokishechnaya plastika zheludka.

The manuscript is devoted to the outstanding Russian surgeon Zakharov E.I.. He first performed gastrojejunoduodenoplasty in 1938 (distal stomach repair with small bowel substitute). Zakharov E.I. developed the method of post-resection stomach repair with jejunum segment interposed between the esophagus and duodenum (esophagojejunoduodenoplasty). Further studies confirmed functional advantage of gastroplasty procedures with preserved duodenal passage.

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.

Muscle synergy for upper limb damping behavior during object transport while walking in healthy young individuals.

Transporting an object during locomotion is one of the most common activities humans perform. Previous studies have shown that continuous and predictive control of grip force, along with the inertial load force of the object, is required to complete this task successfully. Another possible CNS strategy to ensure the dynamic stability of the upper limb is to modify the apparent stiffness and damping via altered muscle activation patterns. In this study, the term damping was used to describe a reduction in upper limb vertical oscillation amplitude to maintain the orientation of the hand-held object. The goal of this study was to identify the neuromuscular strategy for controlling the upper limb during object transport while walking. Three-dimensional kinematic and surface electromyography (EMG) data were recorded from eight, right-handed, healthy young adults who were instructed to walk on a treadmill while carrying an object in their dominant/non-dominant hand, with dominant/non-dominant arm positioning but without an object, and without any object or instructed arm-positioning. EMG recordings from the dominant and non-dominant arms were decomposed separately into underlying muscle synergies using non-negative matrix factorization (NNMF). Results revealed that the dominant arm showed higher damping compared to the non-dominant arm. All muscles showed higher mean levels of activation during object transport except for posterior deltoid (PD), with activation peaks occurring around or slightly before heel contact. The muscle synergy analysis revealed an anticipatory stabilization of the shoulder and elbow joints through a proximal-to-distal muscle activation pattern. These activations appear to play an essential role in maintaining the stability of the carried object in addition to the adjustment of grip force against the perturbations caused by heel contact during walking.

The Effects of Attentional Focus Instructions and Task Difficulty in a Paced Fine Motor Skill.

An external focus of attention is considered superior to an internal focus for learning and performance. However, findings specific to changing the task difficulty are inconsistent. The present study used a reciprocal aiming task to determine the effects of attentional focus on motor performance using speed-accuracy paradigm. We constrained timing to examine how internal and external focus of attention influenced accuracy when task difficulty changes. The results indicated greater accuracy on the right target and greater consistency on both targets for the external focus condition, regardless of task difficulty. Our results uniquely demonstrated how instruction modified a speed-accuracy task.

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.

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.

Thermodynamic Analysis of Fast Stages of Specific Lesion Recognition by DNA Repair Enzymes.

The methodology of determination of the thermodynamic parameters of fast stages of recognition and cleavage of DNA substrates is described for the enzymatic processes catalyzed by DNA glycosylases Fpg and hOGG1 and AP endonuclease APE1 during base excision repair (BER) pathway. For this purpose, stopped-flow pre-steady-state kinetic analysis of tryptophan fluorescence intensity changes in proteins and fluorophores in DNA substrates was performed at various temperatures. This approach made it possible to determine 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. The unified features of mechanism for search and recognition of damaged DNA sites by various enzymes of the BER pathway were discovered.

Thermodynamics of Damaged DNA Binding and Catalysis by Human AP Endonuclease 1.

Apurinic/apyrimidinic (AP) endonucleases play an important role in DNA repair and initiation of AP site elimination. One of the most topical problems in the field of DNA repair is to understand the mechanism of the enzymatic process involving the human enzyme APE1 that provides recognition of AP sites and efficient cleavage of the 5'-phosphodiester bond. In this study, a thermodynamic analysis of the interaction between APE1 and a DNA substrate containing a stable AP site analog lacking the C1' hydroxyl group (F site) was performed. Based on stopped-flow kinetic data at different temperatures, the steps of DNA binding, catalysis, and DNA product release were characterized. The changes in the standard Gibbs energy, enthalpy, and entropy of sequential specific steps of the repair process were determined. The thermodynamic analysis of the data suggests that the initial step of the DNA substrate binding includes formation of non-specific contacts between the enzyme binding surface and DNA, as well as insertion of the amino acid residues Arg177 and Met270 into the duplex, which results in the removal of "crystalline" water molecules from DNA grooves. The second binding step involves the F site flipping-out process and formation of specific contacts between the enzyme active site and the everted 5'-phosphate-2'-deoxyribose residue. It was shown that non-specific interactions between the binding surfaces of the enzyme and DNA provide the main contribution into the thermodynamic parameters of the DNA product release step.