[Show abstract][Hide abstract] ABSTRACT: Here, we report the study of a new multichannel DNA fluorescent base analogue 3-hydroxychromone (3HC) to evaluate its suitability as a fluorescent reporter probe of structural transitions during protein-DNA interactions and its comparison with the current commercially available 2-aminopurine (aPu), pyrrolocytosine (Cpy) and 1,3-diaza-2-oxophenoxazine (tCO). For this purpose, fluorescent base analogues were incorporated into DNA helix on the opposite or on the 5'-side of the damaged nucleoside 5,6-dihydrouridine (DHU), which is specifically recognized and removed by Endonuclease VIII. These fluorophores demonstrated different sensitivities to the DNA helix conformational changes. The highest sensitivity and the most detailed information about the conformational changes of DNA induced by protein binding and processing were obtained using the 3HC probe. The application of this new artificial fluorescent DNA base is a very useful tool for the studies of complex mechanisms of protein-DNA interactions. Using 3HC biosensor, the kinetic mechanism of Endonuclease VIII action was specified.
PLoS ONE 01/2014; 9(6):e100007. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Human 8-oxoguanine DNA glycosylase (hOGG1) is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG). In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van't Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.
PLoS ONE 01/2014; 9(6):e98495. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 8-Oxoguanine-DNA glycosylase (OGG1) removes a pre-mutagenic lesion 8-oxoguanine (oxoGua) from DNA and then nicks the nascent abasic (AP) site by β-elimination. Although the structure of OGG1 bound to damaged DNA is known, the dynamic aspects of oxoGua recognition are not well comprehended. In order to understand the mechanisms of substrate recognition and processing we have constructed OGG1 mutants with the active site occluded by replacement of Cys253, which forms a wall of the base-binding pocket, with bulky leucine or isoleucine. The conformational dynamics of OGG1 mutants was characterized by single-turnover kinetics and stopped-flow kinetics with fluorescent detection. Additionally, the conformational mobility of wild-type and mutant OGG1-substrate complex was assessed using molecular dynamics simulations. Although pocket occlusion distorted the active site and greatly decreased the catalytic activity of OGG1, it did not fully prevent processing of oxoGua and AP sites. Both mutants were notably stimulated in the presence of free 8-bromoguanine, indicating that this base can bind to the distorted OGG1 and facilitate β-elimination. The results agree with the concept of enzyme plasticity, suggesting that the active site of OGG1 is flexible enough to compensate partially for distortions caused by mutation.
Journal of Biological Chemistry 08/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecular dynamics (MD) simulation nowadays is an essential part of biological, chemical, and physical research. There is a vast variety of accurate and high-performance MD software facilitating the task. However, simulations of biopolymers on meaningful time scales always produce large trajectories rarely amenable to manual analysis. Such analysis, and especially meaningful data search and extraction, often becomes a bottleneck of in silico experiment along with actual MD computations. Most of the existing software for analysis of MD simulation results is based on command-line, script-guided processes that require the researchers to have an idea about programming language constructions used, often applied to the one and only product, providing an excessive set of analytic features, but sacrificing ease of use, simplicity, and clarity. In this work, we present an open source, cross-platform, GUI-based program, Molecular Dynamics Trajectory Reader and Analyzer (MDTRA), which may be helpful in addressing such issues. MDTRA is a versatile program and does not require scripting (yet supports it), is able to quickly plot the analysis results, and minimizes RAM requirements (Popov et al., 2012). A key MDTRA feature is the logical organization of data handling and treatment. Our program introduces a convenient way to manage data based on a principle of a re-useable “conveyor”, which delivers results from “streams” (trajectories) through “data sources” to “result collectors.” Each stage is adjustable at any time, causing only the affected data sources to be rebuilt. MDTRA allows users to plot and analyze distances, angles, and forces in the molecule. It also implements trajectory-related search and extraction tools, including determination of meaningful torsions of protein backbone, search for stable hydrogen bonds, building 2D-RMSD diagrams, and massive comparative plotting of DNA parameters. MDTRA proved itself useful in a study of DNA repair enzymes OGG1, Fpg, and MutY. An example of analysis of the mobility of tryptophan residues contributing to fluorescence of E. coli Fpg (observed experimentally by Kuznetsov et al., 2007) is shown in the figure.
[Show abstract][Hide abstract] ABSTRACT: Formamidopyrimidine-DNA glycosylase, Fpg protein from Escherichia coli, initiates base excision repair in DNA by removing a wide variety of oxidized lesions. In this study, we perform thermodynamic analysis of the multi-stage interaction of Fpg with specific DNA-substrates containing 7,8-dihydro-8-oxoguanosine (oxoG), or tetrahydrofuran (THF, an uncleavable abasic site analog) and non-specific (G) DNA-ligand based on stopped-flow kinetic data. Pyrrolocytosine, highly fluorescent analog of the natural nucleobase cytosine, is used to record multi-stage DNA lesion recognition and repair kinetics over a temperature range (10-30°C). The kinetic data were used to obtain the standard Gibbs energy, enthalpy and entropy of the specific stages using van't Hoff approach. The data suggest that not only enthalpy-driven exothermic oxoG recognition, but also the desolvation-accompanied entropy-driven enzyme-substrate complex adjustment into the catalytically active state play equally important roles in the overall process.
Nucleic Acids Research 05/2012; 40(15):7384-92. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Positioning of release factor eRF1 toward adenines and the ribose-phosphate backbone of the UAAA stop signal in the ribosomal decoding site was studied using messenger RNA (mRNA) analogs containing stop signal UAA/UAAA and a photoactivatable cross-linker at definite locations. The human eRF1 peptides cross-linked to these analogs were identified. Cross-linkers on the adenines at the 2nd, 3rd or 4th position modified eRF1 near the conserved YxCxxxF loop (positions 125-131 in the N domain), but cross-linker at the 4th position mainly modified the tripeptide 26-AAR-28. This tripeptide cross-linked also with derivatized 3'-phosphate of UAA, while the same cross-linker at the 3'-phosphate of UAAA modified both the 26-28 and 67-73 fragments. A comparison of the results with those obtained earlier with mRNA analogs bearing a similar cross-linker at the guanines indicates that positioning of eRF1 toward adenines and guanines of stop signals in the 80S termination complex is different. Molecular modeling of eRF1 in the 80S termination complex showed that eRF1 fragments neighboring guanines and adenines of stop signals are compatible with different N domain conformations of eRF1. These conformations vary by positioning of stop signal purines toward the universally conserved dipeptide 31-GT-32, which neighbors guanines but is oriented more distantly from adenines.
Nucleic Acids Research 05/2011; 39(16):7134-46. · 8.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To study positioning of the polypeptide release factor eRF1 toward a stop signal in the ribosomal decoding site, we applied photoactivatable mRNA analogs, derivatives of oligoribonucleotides. The human eRF1 peptides cross-linked to these short mRNAs were identified. Cross-linkers on the guanines at the second, third, and fourth stop signal positions modified fragment 31-33, and to lesser extent amino acids within region 121-131 (the "YxCxxxF loop") in the N domain. Hence, both regions are involved in the recognition of the purines. A cross-linker at the first uridine of the stop codon modifies Val66 near the NIKS loop (positions 61-64), and this region is important for recognition of the first uridine of stop codons. Since the N domain distinct regions of eRF1 are involved in a stop-codon decoding, the eRF1 decoding site is discontinuous and is not of "protein anticodon" type. By molecular modeling, the eRF1 molecule can be fitted to the A site proximal to the P-site-bound tRNA and to a stop codon in mRNA via a large conformational change to one of its three domains. In the simulated eRF1 conformation, the YxCxxxF motif and positions 31-33 are very close to a stop codon, which becomes also proximal to several parts of the C domain. Thus, in the A-site-bound state, the eRF1 conformation significantly differs from those in crystals and solution. The model suggested for eRF1 conformation in the ribosomal A site and cross-linking data are compatible.
[Show abstract][Hide abstract] ABSTRACT: Formamidopyrimidine-DNA glycosylase (Fpg) is responsible for removal of 8-oxoguanine (8-oxoG) and other oxidized purine lesions from DNA and can also excise some oxidatively modified pyrimidines [such as dihydrouracil (DHU)]. Fpg is also specific for a base opposite the lesion, efficiently excising 8-oxoG paired with C but not with A. We have applied stopped-flow kinetics using intrinsic tryptophan fluorescence of the enzyme and fluorescence of 2-aminopurine-labeled DNA to analyze the conformational dynamics of Escherichia coli Fpg during processing of good substrates (8-oxoG.C), poor substrates (8-oxoG.A), and substrates of unclear specificity (such as DHU and 8-oxoG opposite T or G). The analysis of fluorescence traces allows us to conclude that when the enzyme encounters its true substrate, 8-oxoG.C, the complex enters the productive catalytic reaction after approximately 50 ms, partitioning the substrate away from the competing dissociation process, while poor substrates linger in the initial encounter complex for longer. Several intermediate ES complexes were attributed to different structures that exist along the reaction pathway. A likely sequence of events is that the damaged base is first destabilized by the enzyme binding and then everted from DNA, followed by insertion of several amino acid residues into DNA and isomerization of the enzyme into a pre-excision complex. We conclude that rejection of the incorrect substrates occurs mostly at the early stage of formation of the pre-eversion recognition complex, supporting the role of indirect readout in damage recognition.