RESUMO
Human terminal deoxynucleotidyl transferase (TdT) can catalyze template-independent DNA synthesis during the V(D)J recombination and DNA repair through nonhomologous end joining. The capacity for template-independent random addition of nucleotides to single-stranded DNA makes this polymerase useful in various molecular biological applications involving sequential stepwise synthesis of oligonucleotides using modified dNTP. Nonetheless, a serious limitation to the applications of this enzyme is strong selectivity of human TdT toward dNTPs in the order dGTP > dTTP ≈ dATP > dCTP. This study involved molecular dynamics to simulate a potential impact of amino acid substitutions on the enzyme's selectivity toward dNTPs. It was found that the formation of stable hydrogen bonds between a nitrogenous base and amino acid residues at positions 395 and 456 is crucial for the preferences for dNTPs. A set of single-substitution and double-substitution mutants at these positions was analyzed by molecular dynamics simulations. The data revealed two TdT mutants-containing either substitution D395N or substitutions D395N+E456N-that possess substantially equalized selectivity toward various dNTPs as compared to the wild-type enzyme. These results will enable rational design of TdT-like enzymes with equalized dNTP selectivity for biotechnological applications.
Assuntos
DNA Nucleotidilexotransferase , Simulação de Dinâmica Molecular , Humanos , DNA Nucleotidilexotransferase/metabolismo , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/genética , Especificidade por Substrato , Desoxirribonucleotídeos/metabolismo , Desoxirribonucleotídeos/química , Nucleotídeos de Timina/metabolismo , Nucleotídeos de Timina/química , Nucleotídeos de Desoxicitosina/metabolismo , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxiadenina/metabolismo , Nucleotídeos de Desoxiadenina/química , Ligação de Hidrogênio , Nucleotídeos de Desoxiguanina/metabolismo , Nucleotídeos de Desoxiguanina/química , Substituição de AminoácidosRESUMO
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.
Assuntos
Clivagem do DNA , Pyrococcus furiosus , Pyrococcus furiosus/enzimologia , Cinética , Proteínas Arqueais/metabolismo , Proteínas Arqueais/química , Especificidade por Substrato , Conformação de Ácido Nucleico , DNA/metabolismoRESUMO
In the cell, DNA polymerase ß (Polß) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polß can fill DNA gaps formed by other DNA repair enzymes. Single-nucleotide polymorphisms (SNPs) in the POLB gene can affect the enzymatic properties of the resulting protein, owing to possible amino acid substitutions. For many SNP-associated Polß variants, an association with cancer, owing to changes in polymerase activity and fidelity, has been shown. In this work, kinetic analyses and molecular dynamics simulations were used to examine the activity of naturally occurring polymorphic variants G274R, G290C, and R333W. Previously, the amino acid substitutions at these positions have been found in various types of tumors, implying a specific role of Gly-274, Gly-290, and Arg-333 in Polß functioning. All three polymorphic variants had reduced polymerase activity. Two substitutions-G274R and R333W-led to the almost complete disappearance of gap-filling and primer elongation activities, a decrease in the deoxynucleotide triphosphate-binding ability, and a lower polymerization constant, due to alterations of local contacts near the replaced amino acid residues. Thus, variants G274R, G290C, and R333W may be implicated in an elevated level of unrepaired DNA damage.
Assuntos
Substituição de Aminoácidos , DNA Polimerase beta , Simulação de Dinâmica Molecular , Polimorfismo de Nucleotídeo Único , DNA Polimerase beta/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/química , Humanos , Cinética , Reparo do DNA/genética , Nucleotídeos/metabolismo , Nucleotídeos/genéticaRESUMO
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 ß (Polß), 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 Polß 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.
Assuntos
Substituição de Aminoácidos , DNA Polimerase beta , Simulação de Dinâmica Molecular , Polimorfismo de Nucleotídeo Único , DNA Polimerase beta/química , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Humanos , Reparo do DNA , Cinética , Domínio Catalítico , DNA/metabolismo , DNA/genética , DNA/química , Domínios ProteicosRESUMO
Terminal 2'-deoxynucleotidyl transferase (TdT) is a unique enzyme capable of catalysing template-independent elongation of DNA 3' ends during V(D)J recombination. The mechanism controlling the enzyme's substrate specificity, which is necessary for its biological function, remains unknown. Accordingly, in this work, kinetic and mutational analyses of human TdT were performed and allowed to determine quantitative characteristics of individual stages of the enzyme-substrate interaction, which overall may ensure the enzyme's operation either in the distributive or processive mode of primer extension. It was found that conformational dynamics of TdT play an important role in the formation of the catalytic complex. Meanwhile, the nature of the nitrogenous base significantly affected both the dNTP-binding and catalytic-reaction efficiency. The results indicated that neutralisation of the charge and an increase in the internal volume of the active site caused a substantial increase in the activity of the enzyme and induced a transition to the processive mode in the presence of Mg2+ ions. Surrogate metal ions Co2+ or Mn2+ also may regulate the switching of the enzymatic process to the processive mode. Thus, the totality of individual factors affecting the activity of TdT ensures effective execution of its biological function.
Assuntos
DNA Nucleotidilexotransferase , DNA Polimerase Dirigida por DNA , Humanos , Especificidade por Substrato , Catálise , Corantes , Nucleotídeos , ÍonsRESUMO
Coordination of enzymatic activities in the course of base excision repair (BER) is essential to ensure complete repair of damaged bases. Two major mechanisms underlying the coordination of BER are known today: the "passing the baton" model and a model of preassembled stable multiprotein repair complexes called "repairosomes." In this work, we aimed to elucidate the coordination between human apurinic/apyrimidinic (AP) endonuclease APE1 and DNA polymerase Polß in BER through studying an impact of APE1 on Polß-catalyzed nucleotide incorporation into different model substrates that mimic different single-strand break (SSB) intermediates arising along the BER pathway. It was found that APE1's impact on separate stages of Polß's catalysis depends on the nature of a DNA substrate. In this complex, APE1 removed 3' blocking groups and corrected Polß-catalyzed DNA synthesis in a coordinated manner. Our findings support the hypothesis that Polß not only can displace APE1 from damaged DNA within the "passing the baton" model but also performs the gap-filling reaction in the ternary complex with APE1 according to the "repairosome" model. Taken together, our results provide new insights into coordination between APE1 and Polß during the BER process.
Assuntos
DNA Polimerase beta , Humanos , DNA Polimerase beta/metabolismo , Reparo do DNA , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Complexos Multiproteicos , DNA/química , Endonucleases/genética , Endonucleases/metabolismoRESUMO
DNA-dependent DNA polymerases have been intensively studied for more than 60 years and underlie numerous biotechnological and diagnostic applications. In vitro, DNA polymerases are used for DNA manipulations, including cloning, PCR, site-directed mutagenesis, sequencing, and others. Understanding the mechanisms of action of DNA polymerases is important for the creation of new enzymes possessing improved or modified properties. This review is focused on archaeal family B DNA polymerases. These enzymes have high fidelity and thermal stability and are finding many applications in molecular biological methods. Nevertheless, the search for and construction of new DNA polymerases with altered properties is constantly underway, including enzymes for synthetic biology. This brief review describes advances in the development of family B DNA polymerases for PCR, synthesis of xeno-nucleic acids, and reverse transcription.
RESUMO
Introduction: Detrended Fluctuation Analysis (DFA) has been used to investigate self-similarity in center of pressure (CoP) time series. For fractional gaussian noise (fGn) signals, the analysis returns a scaling exponent, DFA-α, whose value characterizes the temporal correlations as persistent, random, or anti-persistent. In the study of postural control, DFA has revealed two time scaling regions, one at the short-term and one at the long-term scaling regions in the diffusion plots, suggesting different types of postural dynamics. Much attention has been given to the selection of minimum and maximum scales, but the choice of spacing (step size) between the window sizes at which the fluctuation function is evaluated may also affect the estimates of scaling exponents. The aim of this study is twofold. First, to determine whether DFA can reveal postural adjustments supporting performance of an upper limb task under variable demands. Second, to compare evenly-spaced DFA with two different step sizes, 0.5 and 1.0 in log2 units, applied to CoP time series. Methods: We analyzed time series of anterior-posterior (AP) and medial-lateral (ML) CoP displacement from healthy participants performing a sequential upper limb task under variable demand. Results: DFA diffusion plots revealed two scaling regions in the AP and ML CoP time series. The short-term scaling region generally showed hyper-diffusive dynamics and long-term scaling revealed mildly persistent dynamics in the ML direction and random-like dynamics in the AP direction. There was a systematic tendency for higher estimates of DFA-α and lower estimates for crossover points for the 0.5-unit step size vs. 1.0-unit size. Discussion: Results provide evidence that DFA-α captures task-related differences between postural adjustments in the AP and ML directions. Results also showed that DFA-α estimates and crossover points are sensitive to step size. A step size of 0.5 led to less variable DFA-α for the long-term scaling region, higher estimation for the short-term scaling region, lower estimate for crossover points, and revealed anomalous estimates at the very short range that had implications for choice of minimum window size. We, therefore, recommend the use of 0.5 step size in evenly spaced DFAs for CoP time series similar to ours.
RESUMO
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.
Assuntos
Aminoácidos , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Aminoácidos/genética , Especificidade por Substrato , Cinética , Espectroscopia de Ressonância de Spin Eletrônica , Dano ao DNA , Reparo do DNA , DNA/química , Endonucleases/metabolismo , Nucleotídeos , DesoxiuridinaRESUMO
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.
Assuntos
Dioxigenases , Humanos , Dioxigenases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Especificidade por Substrato , DNA/metabolismo , AminoácidosRESUMO
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 ß, 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 Polß'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 Polß 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 Polß's activity toward the model DNA intermediates.
Assuntos
DNA Glicosilases , DNA Polimerase beta , Humanos , DNA Polimerase beta/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Glicosilases/metabolismo , Replicação do DNA , DNA , Dano ao DNARESUMO
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 ß (Polß), 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 Polß, 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 Polß catalytic activity. Thus, a decrease in the activity of studied mutant forms may be associated with an increased risk of damage to the genome.
Assuntos
DNA Polimerase beta , Transferases , Humanos , Substituição de Aminoácidos , DNA/metabolismo , DNA Polimerase beta/genética , DNA Polimerase beta/metabolismo , Reparo do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Transferases/genética , Transferases/metabolismoRESUMO
Adopting an external focus of attention (EF) has been found beneficial over internal focus (IF) for performing motor skills. Previous studies primarily examined focus of attention (FOA) effects on performance outcomes (such as error and accuracy), with relatively less emphasis on movement coordination. Given that human movements are kinematically and kinetically abundant (Gefland & Latash, 1998), FOA instructions may change how motor abundance is utilized by the CNS. This study applied the uncontrolled manifold analysis (UCM) to address this question in a reaching task. Healthy young adults (N = 38; 22 ± 1 yr; 7 men, 31 women) performed planar reaching movements to a target using either the dominant or nondominant arm under two different FOA instructions: EF and IF. Reaching was performed without online visual feedback and at a preferred pace. Joint angles of the clavicle-scapula, shoulder, elbow, and wrist were recorded, and their covariation for controlling dowel endpoint position was analyzed via UCM. As expected, IF led to a higher mean radial error than EF, driven by increases in aiming bias and variability. Consistent with this result, the UCM analysis showed that IF led to higher goal-relevant variance among the joints (VORT) compared to EF starting from the first 20% of the reach to the end. However, the goal-irrelevant variance (VUCM)-index of joint variance that does not affect the end-effector position-did not show FOA effects. The index of stability of joint coordination with respect to endpoint position (ΔV) was also not different between the EF and IF. Consistent with the constrained action hypothesis, these results provide evidence that IF disrupted goal-relevant joint covariation starting in the early phases of the reach without affecting goal-irrelevant coordination.
Assuntos
Articulação do Cotovelo , Masculino , Adulto Jovem , Humanos , Feminino , Movimento , Ombro , Extremidade Superior , Atenção , Fenômenos Biomecânicos , Desempenho PsicomotorRESUMO
Since the discovery of anticancer properties of a naturally occurring hexacyclic marine alkaloid Lamellarin D, the attempts have been made to prepare its synthetic analogues and elucidate the effects of each structural component on their activity profile. While F-ring-free, A-ring-free and B-ring-open lamellarins are known, E-ring-free analogues have never been investigated. In this work, we developed a facile and straightforward synthetic method toward E-ring-free lamellarin analogues based on the [3+2]-cycloaddition. For the first time, we prepared several pentacyclic lamellarin analogues without E-ring in their structure and assessed their cytotoxicity in a panel of cancer cell lines in comparison with several hexacyclic lamellarins. E-ring-free lamellarins were devoid of cytotoxicity due to their poor solubility in cellular environment.
Assuntos
Alcaloides , Antineoplásicos , Neoplasias , Humanos , Antineoplásicos/química , Neoplasias/tratamento farmacológico , Alcaloides/química , Linhagem Celular , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Cumarínicos/química , Relação Estrutura-AtividadeRESUMO
DNA polymerase ß (Polß) expression is essential for the cell's response to DNA damage that occurs during natural cellular processes. Polß is considered the main reparative DNA polymerase, whose role is to fill the DNA gaps arising in the base excision repair pathway. Mutations in Polß can lead to cancer, neurodegenerative diseases, or premature aging. Many single-nucleotide polymorphisms have been identified in the POLB gene, but the consequences of these polymorphisms are not always clear. It is known that some polymorphic variants in the Polß sequence reduce the efficiency of DNA repair, thereby raising the frequency of mutations in the genome. In the current work, we studied two polymorphic variants (G118V and R149I separately) of human Polß that affect its DNA-binding region. It was found that each amino acid substitution alters Polß's affinity for gapped DNA. Each polymorphic variant also weakens its binding affinity for dATP. The G118V variant was found to greatly affect Polß's ability to fill gapped DNA and slowed the catalytic rate as compared to the wild-type enzyme. Thus, these polymorphic variants seem to decrease the ability of Polß to maintain base excision repair efficiency.
Assuntos
Dano ao DNA , Reparo do DNA , Humanos , Catálise , DNA/metabolismo , Reparo do DNA/genética , Polimorfismo de Nucleotídeo Único , Especificidade por Substrato , BiocatáliseRESUMO
The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product-enzyme complex by the next enzyme in the pathway, resulting in the "passing-the-baton" model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1-DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme-product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polß stimulates both i) APE1 binding to an abasic-site-containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polß activities and suggest that Polß is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER.
Assuntos
DNA Polimerase beta , Humanos , DNA/metabolismo , Dano ao DNA , DNA Polimerase beta/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismoRESUMO
Neuromotor dysfunction after a concussion is common, but balance tests used to assess neuromotor dysfunction are typically subjective. Current objective balance tests are either cost- or space-prohibitive, or utilize a static balance protocol, which may mask neuromotor dysfunction due to the simplicity of the task. To address this gap, our team developed an Android-based smartphone app (portable and cost-effective) that uses the sensors in the device (objective) to record movement profiles during a stepping-in-place task (dynamic movement). The purpose of this study was to examine the extent to which our custom smartphone app and protocol could discriminate neuromotor behavior between concussed and non-concussed participants. Data were collected at two university laboratories and two military sites. Participants included civilians and Service Members (N = 216) with and without a clinically diagnosed concussion. Kinematic and variability metrics were derived from a thigh angle time series while the participants completed a series of stepping-in-place tasks in three conditions: eyes open, eyes closed, and head shake. We observed that the standard deviation of the mean maximum angular velocity of the thigh was higher in the participants with a concussion history in the eyes closed and head shake conditions of the stepping-in-place task. Consistent with the optimal movement variability hypothesis, we showed that increased movement variability occurs in participants with a concussion history, for which our smartphone app and protocol were sensitive enough to capture.
Assuntos
Concussão Encefálica , Militares , Aplicativos Móveis , Humanos , Concussão Encefálica/diagnóstico , Fenômenos Biomecânicos , Extremidade Inferior , Smartphone , Equilíbrio PosturalRESUMO
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' â 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.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos) , Proteínas de Saccharomyces cerevisiae , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Saccharomyces cerevisiae/metabolismo , Cinética , Endonucleases , ExonucleasesRESUMO
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.
Assuntos
DNA Glicosilases , DNA Liase (Sítios Apurínicos ou Apirimidínicos) , DNA/metabolismo , Dano ao DNA , DNA Glicosilases/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/genética , Escherichia coli/metabolismo , Exonucleases/genética , Humanos , NucleotídeosRESUMO
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.