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Apurinic/apyrimidinic endonuclease 1 (APE1) is responsible for the hydrolysis of the phosphodiester bond on the 5′ side of an apurinic/apyrimidinic site during base excision repair. Moreover, in DNA, this enzyme can recognize nucleotides containing such damaged bases as 5,6-dihydro-2′-deoxyuridine (DHU), 2′-deoxyuridine (dU), alpha-2′-deoxyadenosine (αA), and 1,N6-ethenoadenosine (εA). Previously, by pulsed electron–electron double resonance spectroscopy and pre-steady-state kinetic analysis, we have revealed multistep DNA rearrangements during the formation of the catalytic complex. In the present study, the modeling of the eversion trajectory of nucleotides with various damaged bases was performed by directed molecular dynamics simulations. It was found that each damaged base at the beginning of the eversion interacts with protein loop Val196-Arg201, which should be moved to enable further nucleotide eversion. This movement involves a shift in loop Val196-Arg201 away from loop Asn253-Thr257 and requires the disruption of contacts between these loops. The Glu260Ala substitution facilitates the separation of the two loops. Moreover, conformational changes in the Asn253-Thr257 loop should occur in the second half of the lesion eversion trajectory. All these perturbations within the protein globule tend to reduce steric interactions of each damaged base with the protein during the eversion of the nucleotide from DNA and movement to the active site. These perturbations are important determinants of substrate specificity of endonuclease APE1. [ABSTRACT FROM AUTHOR]

R-loops can act as replication fork barriers, creating transcription–replication collisions and inducing replication stress by arresting DNA synthesis, thereby possibly causing aberrant processing and the formation of DNA strand breaks. RNase H1 (RH1) is one of the enzymes that participates in R-loop degradation by cleaving the RNA strand within a hybrid RNA–DNA duplex. In this study, the kinetic features of the interaction of RH1 from Escherichia coli with R-loops of various structures were investigated. It was found that the values of the dissociation constants Kd were minimal for complexes of RH1 with model R-loops containing a 10–11-nt RNA–DNA hybrid part, indicating effective binding. Analysis of the kinetics of RNA degradation in the R-loops by RH1 revealed that the rate-limiting step of the process was catalytic-complex formation. In the presence of RNA polymerase, the R-loops containing a ≤16-nt RNA–DNA hybrid part were efficiently protected from cleavage by RH1. In contrast, R-loops containing longer RNA–DNA hybrid parts, as a model of an abnormal transcription process, were not protected by RNA polymerase and were effectively digested by RH1. [ABSTRACT FROM AUTHOR]

The mechanism of transcription proceeds through the formation of R-loop structures containing a DNA–RNA heteroduplex and a single-stranded DNA segment that should be placed inside the elongation complex; therefore, these nucleic acid segments are limited in length. The attachment of each nucleotide to the 3′ end of an RNA strand requires a repeating cycle of incoming nucleoside triphosphate binding, catalysis, and enzyme translocation. Within these steps of transcription elongation, RNA polymerase sequentially goes through several states and is post-translocated, catalytic, and pre-translocated. Moreover, the backward movement of the polymerase, which is essential for transcription pausing and proofreading activity, gives rise to a backtracked state. In the present study, to analyze both the efficacy of transcription elongation complex (TEC) formation and the rate of RNA synthesis, we used a set of model R-loops that mimic the pre-translocated state, post-translocated state, backtracked state, and a misincorporation event. It was shown that TEC assembly proceeds as an equilibrium process, including the simultaneous formation of a catalytically competent TEC as well as a catalytically inactive conformation. Our data suggest that the inactive complex of RNA polymerase with an R-loop undergoes slow conformational changes, resulting in a catalytically competent TEC. It was revealed that the structural features of R-loops affect the ratio between active and inactive states of the TEC, the rate of conformational rearrangements required for the induced-fit transition from the inactive state to the catalytically competent TEC, and the rates of accumulation of both the total RNA products and long RNA products. [ABSTRACT FROM AUTHOR]

This review deals with the functional characteristics and biological roles of enzymes participating in DNA methylation and demethylation as key factors in epigenetic regulation of gene expression. The set of enzymes that carry out such processes in human cells is limited to representatives of two families, namely DNMT (DNA methyltransferases) and TET (DNA dioxygenases). The review presents detailed information known today about each functionally important member of these families and describes the catalytic activity and roles in the mammalian body while also providing examples of dysregulation of the expression and/or activity of these enzymes in conjunction with the development of some human disorders, including cancers, neurodegenerative diseases, and developmental pathologies. By combining the up-to-date information on the dysfunction of various enzymes that control the DNA "methylome" in the human body, we hope not only to draw attention to the importance of the maintenance of a required DNA methylation level (ensuring epigenetic regulation of gene expression and normal functioning of the entire body) but also to help identify new targets for directed control over the activity of the enzymes that implement the balance between processes of DNA methylation and demethylation. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs) are widespread, persistent, and toxic environmental pollutants. Many anthropogenic and some natural factors contribute to the spread and accumulation of PAHs in aquatic and soil systems. The effective and environmentally friendly remediation of these chemical compounds is an important and challenging problem that has kept scientists busy over the last few decades. This review briefly summarizes data on the main sources of PAHs, their toxicity to living organisms, and physical and chemical approaches to the remediation of PAHs. The basic idea behind existing approaches to the bioremediation of PAHs is outlined with an emphasis on a detailed description of the use of bacterial strains as individual isolates, consortia, or cell-free enzymatic agents. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A chemical modification of cellulose diacetate by phthalate and nitrate was performed to increase solubility in organic solvents and change the electrical properties. The role of substituents on the conductivity, permittivity, and polarizability of cellulose films is revealed. It has been shown that highly porous micro particles can be obtained from cellulose derivatives by a simple and technological freeze‐drying method. The resulting micro sized aerogels have a predominantly spherical morphology and amorphous structure. Suspensions of porous particles of nitro‐ and phthalylated cellulose derivatives in silicone oil have an increased dielectric permittivity compared to cellulose diacetate particles. Produced particles are novel promising material with tunable electrical properties for advanced applications in composites, including for electrorheological fluids. [ABSTRACT FROM AUTHOR]

In the cell, DNA polymerase β (Polβ) is involved in many processes aimed at maintaining genome stability and is considered the main repair DNA polymerase participating in base excision repair (BER). Polβ 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 Polβ 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 Polβ 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. [ABSTRACT FROM AUTHOR]

Mau DNA polymerase is a family A DNA polymerase isolated from Massilia aurea. In this study, a recombinant plasmid, His6-tagged Mau-pET28c, was constructed. His-tagged Mau was expressed in Escherichia coli Rosseta 2 (DE3) competent cells and, after optimization of purification conditions, was successfully isolated via a two-step purification system by Ni2+-chelating affinity chromatography followed by heparin affinity chromatography. The biochemical properties of Mau DNA polymerase were investigated next. This polymerase showed maximal polymerase activity at 30 °C, pH 8.4–8.8, 2–10 mM MgCl2, and 10–40 mM KCl. Kinetic parameters of correct and incorrect dNTP incorporation as well as DNA-binding affinity were determined too. KdNTPd,app values were found to be 16 µM for correct dNTP and 200–500 µM for incorrect dNTP. The kinetic parameter kcat turned out to be 0.2 s−1 for correct dNTP incorporation and an order of magnitude less for incorrect dNTP incorporation. It was demonstrated that Mau DNA polymerase has 5′→3′ and 3′→5′ exonuclease activities associated with the main activity. [ABSTRACT FROM AUTHOR]