SNP-Associated Substitutions of Amino Acid Residues in the dNTP Selection Subdomain Decrease Polß Polymerase Activity.

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.

The Impact of SNP-Induced Amino Acid Substitutions L19P and G66R in the dRP-Lyase Domain of Human DNA Polymerase ß on Enzyme Activities.

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.

Substrate Specificity Diversity of Human Terminal Deoxynucleotidyltransferase May Be a Naturally Programmed Feature Facilitating Its Biological Function.

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.

Coordination between human DNA polymerase ß and apurinic/apyrimidinic endonuclease 1 in the course of DNA repair.

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.

Direct Enzyme Engineering of B Family DNA Polymerases for Biotechnological Approaches.

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.

Individual Contributions of Amido Acid Residues Tyr122, Ile168, and Asp173 to the Activity and Substrate Specificity of Human DNA Dioxygenase ABH2.

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.

Inner Amino Acid Contacts Are Key Factors of Multistage Structural Rearrangements of DNA and Affect Substrate Specificity of Apurinic/Apyrimidinic Endonuclease APE1.

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.

The Impact of Human DNA Glycosylases on the Activity of DNA Polymerase ß toward Various Base Excision Repair Intermediates.

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.

The Activity of Natural Polymorphic Variants of Human DNA Polymerase ß Having an Amino Acid Substitution in the Transferase Domain.

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.

Human Polß Natural Polymorphic Variants G118V and R149I Affects Substate Binding and Catalysis.

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.

Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase ß on the APE1-DNA interaction.

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.

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.

The Kinetic Mechanism of 3'-5' Exonucleolytic Activity of AP Endonuclease Nfo from E. coli.

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.

Insight into the mechanism of DNA synthesis by human terminal deoxynucleotidyltransferase.

Terminal deoxynucleotidyltransferase (TdT) is a member of the DNA polymerase X family that is responsible for random addition of nucleotides to single-stranded DNA. We present investigation into the role of metal ions and specific interactions of dNTP with active-site amino acid residues in the mechanisms underlying the recognition of nucleoside triphosphates by human TdT under pre-steady-state conditions. In the elongation mode, the ratios of translocation and dissociation rate constants, as well as the catalytic rate constant were dependent on the nature of the nucleobase. Preferences of TdT in dNTP incorporation were researched by molecular dynamics simulations of complexes of TdT with a primer and dNTP or with the elongated primer. Purine nucleotides lost the "summarised" H-bonding network after the attachment of the nucleotide to the primer, whereas pyrimidine nucleotides increased the number and relative lifetime of H-bonds in the post-catalytic complex. The effect of divalent metal ions on the primer elongation revealed that Me²⁺ cofactor can significantly change parameters of the primer elongation by strongly affecting the rate of nucleotide attachment and the polymerisation mode.

The mechanism of damage recognition by apurinic/apyrimidinic endonuclease Nfo from Escherichia coli.

Apurinic/apyrimidinic (AP) endonuclease Nfo from Escherichia coli recognises AP sites in DNA and catalyses phosphodiester bond cleavage on the 5' side of AP sites and some damaged or undamaged nucleotides. Here, the mechanism of target nucleotide recognition by Nfo was analysed by pulsed electron-electron double resonance (PELDOR, also known as DEER) spectroscopy and pre-steady-state kinetic analysis with Förster resonance energy transfer detection of DNA conformational changes during DNA binding. The efficiency of endonucleolytic cleavage of a target nucleotide in model DNA substrates was ranked as (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran [F-site] > 5,6-dihydro-2'-deoxyuridine > α-anomer of 2'-deoxyadenosine >2'-deoxyuridine > undamaged DNA. Real-time conformational changes of DNA during interaction with Nfo revealed an increase of distances between duplex ends during the formation of the initial enzyme-substrate complex. The use of rigid-linker spin-labelled DNA duplexes in DEER measurements indicated that double-helix bending and unwinding by the target nucleotide itself is one of the key factors responsible for indiscriminate recognition of a target nucleotide by Nfo. The results for the first time show that AP endonucleases from different structural families utilise a common strategy of damage recognition, which globally may be integrated with the mechanism of searching for specific sites in DNA by other enzymes.

Analyses of pre-steady-state kinetics and isotope effects of the γ-elimination reaction catalyzed by Citrobacter freundii methionine γ-lyase.

Methionine γ-lyase (MGL) is a pyridoxal 5'-phosphate-dependent enzyme catalyzing γ-elimination in l-methionine. Pyridoxal 5'-phosphate-dependent enzymes have unique spectral properties that allow to monitor sequential formation and decomposition of various intermediates via the detection of absorbance changes. The kinetic mechanism of the γ-elimination reaction catalyzed by Citrobacter freundii MGL was elucidated here by fast stopped-flow kinetic analysis. Single-wavelength detection of characteristic absorbance changes enabled us to compare transformations of intermediates in the course of the reaction with different substrates. The influence of various γ-substituents in the substrate on the formation of key intermediates was estimated. Kinetic isotope effects of α- and ß-protons were determined using deuterium-substituted l-methionine. Contributions of amino acid residues Tyr113 and Tyr58 located in the active site on the formation and decomposition of reaction intermediates were identified too. α-Aminocrotonate formation is the rate-limiting step of the enzymatic γ-elimination reaction. Kinetic isotope effects strongly support concerted reaction mechanisms of transformation between an external aldimine and a ketimine intermediate as well as a ketimine intermediate and an unsaturated ketimine.

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.

Structural and Functional Differences between Homologous Bacterial Ribonucleases.

Small cationic guanyl-preferring ribonucleases (RNases) produced by the Bacillus species share a similar protein tertiary structure with a high degree of amino acid sequence conservation. However, they form dimers that differ in conformation and stability. Here, we have addressed the issues (1) whether the homologous RNases also have distinctions in catalytic activity towards different RNA substrates and interactions with the inhibitor protein barstar, and (2) whether these differences correlate with structural features of the proteins. Circular dichroism and dynamic light scattering assays revealed distinctions in the structures of homologous RNases. The activity levels of the RNases towards natural RNA substrates, as measured spectrometrically by acid-soluble hydrolysis products, were similar and decreased in the row high-polymeric RNA >>> transport RNA > double-stranded RNA. However, stopped flow kinetic studies on model RNA substrates containing the guanosine residue in a hairpin stem or a loop showed that the cleavage rates of these enzymes were different. Moreover, homologous RNases were inhibited by the barstar with diverse efficiency. Therefore, minor changes in structure elements of homologous proteins have a potential to significantly effect molecule stability and functional activities, such as catalysis or ligand binding.

Modified covariance beamformer for solving MEG inverse problem in the environment with correlated sources.

Magnetoencephalography (MEG) is a neuroimaging method ideally suited for non-invasive studies of brain dynamics. MEG's spatial resolution critically depends on the approach used to solve the ill-posed inverse problem in order to transform sensor signals into cortical activation maps. Over recent years non-globally optimized solutions based on the use of adaptive beamformers (BF) gained popularity. When operating in the environment with a small number of uncorrelated sources the BFs perform optimally and yield high spatial resolution. However, the BFs are known to fail when dealing with correlated sources acting like poorly tuned spatial filters with low signal-to-noise ratio (SNR) of the output timeseries and often meaningless cortical maps of power distribution. This fact poses a serious limitation on the broader use of this promising technique especially since fundamental mechanisms of brain functioning, its inherent symmetry and task-based experimental paradigms result into a great deal of correlation in the activity of cortical sources. To cope with this problem, we developed a novel data covariance modification approach that allows for building beamformers that maintain high spatial resolution when operating in the environments with correlated sources. At the core of our method is a projection operation applied to the vectorized sensor-space covariance matrix. This projection does not remove the activity of the correlated sources from the sensor-space covariance matrix but rather selectively handles their contributions to the covariance matrix and creates a sufficiently accurate approximation of an ideal data covariance that could hypothetically be observed should these sources be uncorrelated. Since the projection operation is reciprocal to the PSIICOS method developed by us earlier (Ossadtchi et al., 2018) we refer to the family of algorithms presented here as ReciPSIICOS. We assess the performance of the novel approach using realistically simulated MEG data and show its superior performance in comparison to the classical BF approaches and well established MNE as a method immune to source synchrony by design. We have also applied our approach to the MEG datasets from the two experiments involving two different auditory tasks. The analysis of experimental MEG datasets showed that beamformers from ReciPSIICOS family, but not the classical BF, discovered the expected bilateral focal sources in the primary auditory cortex and detected motor cortex activity associated with the audio-motor task. In most cases MNE managed well but as expected produced more spatially diffuse source distributions. Notably, ReciPSIICOS beamformers yielded cortical activity estimates with SNR several times higher than that obtained with the classical BF, which may indirectly indicate the severeness of the signal cancellation problem when applying classical beamformers to MEG signals generated by synchronous sources.

Cortical plasticity elicited by acoustically cued monetary losses: an ERP study.

Both human and animal studies have demonstrated remarkable findings of experience-induced plasticity in the cortex. Here, we investigated whether the widely used monetary incentive delay (MID) task changes the neural processing of incentive cues that code expected monetary outcomes. We used a novel auditory version of the MID task, where participants responded to acoustic cues that coded expected monetary losses. To investigate task-induced brain plasticity, we presented incentive cues as deviants during passive oddball tasks before and after two sessions of the MID task. During the oddball task, we recorded the mismatch-related negativity (MMN) as an index of cortical plasticity. We found that two sessions of the MID task evoked a significant enhancement of MMN for incentive cues that predicted large monetary losses, specifically when monetary cue discrimination was essential for maximising monetary outcomes. The task-induced plasticity correlated with the learning-related neural activity recorded during the MID task. Thus, our results confirm that the processing of (loss)incentive auditory cues is dynamically modulated by previously learned monetary outcomes.