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Covalent protein capture (cross-linking) by reactive DNA derivatives makes it possible to investigate structural features by fixing complexes at different stages of DNA–protein recognition. The most common cross-linking methods are based on reactive groups that interact with native or engineered cysteine residues. Nonetheless, high reactivity of most of such groups leads to preferential fixation of early-stage complexes or even non-selective cross-linking. We synthesised a set of DNA reagents carrying an acrylamide group attached to the C5 atom of a 2′-deoxyuridine moiety via various linkers and studied cross-linking with MutS as a model protein. MutS scans DNA for mismatches and damaged nucleobases and can form multiple non-specific complexes with DNA that may cause non-selective cross-linking. By varying the length of the linker between DNA and the acrylamide group and by changing the distance between the reactive nucleotide and a mismatch in the duplex, we showed that cross-linking occurs only if the distance between the acrylamide group and cysteine is optimal within the DNA–protein complex. Thus, acrylamide-modified DNA duplexes are excellent tools for studying DNA–protein interactions because of high selectivity of cysteine trapping. [ABSTRACT FROM AUTHOR]

6S RNA, a small non-coding RNA present in almost all bacteria, inhibits transcription via direct binding to RNA polymerase holoenzymes. The mechanism of 6S RNA action was investigated to a large extent in E. coli, however, lack of 6S RNA (ΔssrS) was demonstrated to be unfavorable but not essential for cell survival under various growth conditions. In the present study, we revealed, for the first time, a lethal phenotype of the ΔssrS strain in the presence of high concentrations of H2O2. This phenotype was rescued by complementation of the ssrS gene on a plasmid. We performed comparative qRT-PCR analyses on an enlarged set of mRNAs of genes associated with the oxidative stress response, allowing us to identify four genes known to be involved in this pathway (soxS, ahpC, sodA and tpx) that had decreased mRNA levels in the ΔssrS strain. Finally, we performed comparative proteomic analyses of the wild-type and ΔssrS strains, confirming that ΔssrS bacteria have reduced levels of the proteins AhpC and Tpx involved in H2O2 reduction. Our findings substantiate the crucial role of the riboregulator 6S RNA for bacterial coping with extreme stresses. [ABSTRACT FROM AUTHOR]

Various studies are conducted to determine the ability of the various [beta] and [lamda] DNA polymerases to bypass the thymine glycol (Tg) in the gapped DNA structures. The DNA polymerase [lamda] is shown to be extremely beneficial in catalyzing the DNA synthesis across Tg.

Nicking endonucleases (NEases) selectively cleave single DNA strands in double-stranded DNAs at a specific site. They are widely used in bioanalytical applications and in genome editing; however, the peculiarities of DNA–protein interactions for most of them are still poorly studied. Previously, it has been shown that the large subunit of heterodimeric restriction endonuclease BspD6I (Nt.BstD6I) acts as a NEase. Here we present a study of interaction of restriction endonuclease BspD6I with modified DNA containing single non-nucleotide insertion with an azobenzene moiety in the enzyme cleavage sites or in positions of sugar-phosphate backbone nearby. According to these data, we designed a number of effective stimulus-responsive oligonucleotide inhibitors bearing azobenzene or triethylene glycol residues. These modified oligonucleotides modulated the functional activity of Nt.BspD6I after cooling or heating. We were able to block the cleavage of T7 phage DNA by this enzyme in the presence of such inhibitors at 20–25°C, whereas the Nt.BspD6I ability to hydrolyze DNA was completely restored after heating to 45°C. The observed effects can serve as a basis for the development of a platform for regulation of NEase activity in vitro or in vivo by external signals. [ABSTRACT FROM AUTHOR]

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active “sliding clamp” conformation on Tg-DNA is problematic. [ABSTRACT FROM AUTHOR]

In this work, the possibility of constructing a thermo-switchable enzyme according to the 'molecular gate' strategy is demonstrated. The approach is based on the covalent attachment of oligodeoxyribonucleotides to cysteine residues of an enzyme adjacent to its active center to form a temporal barrier for enzyme-substrate complex formation. The activity of the modified enzyme that had been studied here-the restriction endonuclease SsoII (R.SsoII)-was diminished by a factor of 180 at 25 °С that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 °С resulted in a 30-fold increase of activity. The activity of unmodified R.SsoII also increased on heating from 25 to 45 °; however, the difference did not exceed a factor of 3-4. The changes in enzymatic activity observed were shown to be reversible for both the unmodified and the modified R.SsoII. Variation of the length and the sequence of the attached oligodeoxyribonucleotides might allow greater modulation of the activity of DNA-protein conjugates. © 2013 IUBMB Life, 65(12):1012-1016, 2013 [ABSTRACT FROM AUTHOR]

Lesions in the DNA arise under ionizing irradiation conditions or various chemical oxidants as a single damage or as part of a multiply damaged site within 1–2 helical turns (clustered lesion). Here, we explored the repair opportunity of the apurinic/apyrimidinic site (AP site) composed of the clustered lesion with 5-formyluracil (5-foU) by the base excision repair (BER) proteins. We found, that if the AP site is shifted relative to the 5-foU of the opposite strand, it could be repaired primarily via the short-patch BER pathway. In this case, the cleavage efficiency of the AP site-containing DNA strand catalyzed by human apurinic/apyrimidinic endonuclease 1 (hAPE1) decreased under AP site excursion to the 3'-side relative to the lesion in the other DNA strand. DNA synthesis catalyzed by DNA polymerase lambda was more accurate in comparison to the one catalyzed by DNA polymerase beta. If the AP site was located exactly opposite 5-foU it was expected to switch the repair to the long-patch BER pathway. In this situation, human processivity factor hPCNA stimulates the process. [ABSTRACT FROM AUTHOR]

Nicking endonucleases (NEs) are enzymes that incise only one strand of the duplex to produce a DNA molecule that is 'nicked' rather than cleaved in two. Since these precision tools are used in genetic engineering and genome editing, information about their mechanism of action at all stages of DNA recognition and phosphodiester bond hydrolysis is essential. For the first time, fast kinetics of the Nt.BspD6I interaction with DNA were studied by the stopped-flow technique, and changes of optical characteristics were registered for the enzyme or DNA molecules. The role of divalent metal cations was estimated at all steps of Nt.BspD6I–DNA complex formation. It was demonstrated that divalent metal ions are not required for the formation of a non-specific complex of the protein with DNA. Nt.BspD6I bound five-fold more efficiently to its recognition site in DNA than to a random DNA. DNA bending was confirmed during the specific binding of Nt.BspD6I to a substrate. The optimal size of Nt.BspD6I's binding site in DNA as determined in this work should be taken into account in methods of detection of nucleic acid sequences and/or even various base modifications by means of NEs. [ABSTRACT FROM AUTHOR]

Abstract: In order to better understand the adsorption mechanism of nucleic acids at electrode surface, MAC Mode atomic force microscopy (MAC Mode AFM) was used to investigate the adsorption of a 10-base synthetic oligodeoxynucleotide (ODN) with the sequence 5′-CTTTTTCTTT-3′ containing only pyrimidinic bases, onto a highly oriented pyrolytic graphite (HOPG) electrode. Free adsorption and adsorption at applied potentials of +0.30 and +0.65V versus AgQRE, were carried out. AFM images in air demonstrated that the molecules adsorb spontaneously on the electrode surface. The ODNs have the tendency to self-assemble from solution onto the solid support in a tight and well-spread two-dimensional lattice covering the entire surface, showing the existence of different molecular conformations and exposing parts of the HOPG surface. The degree of surface coverage and the adsorption pattern were directly dependent on ODN concentration and immobilization procedure. During free adsorption, the hydrophobic interactions of the ODNs with the HOPG represent the main adsorption mechanism. When a positive potential is applied to the HOPG, electrostatic interactions between the positively charged electrode surface and the negatively charged sugar-phosphate backbones of ODNs are predominant. [Copyright &y& Elsevier]

The preparation of two novel 2′-O-alkyl phosphoramidites bearing 1,2-diol and hydrazide functions for a chemoselective ligation is described. The former amidite was used to obtain 2′-modified oligodeoxyribonucleotides, which can be later oxidised by NaIO4 to generate 2′-aldehyde oligonucleotides. These were successfully conjugated to acceptor molecules. The latter amidite also showed good coupling yields, but the hydrazide function was demonstrated to be labile under basic deprotection conditions. [ABSTRACT FROM AUTHOR]

The double-stranded oligodeoxyribonucleotides with single internucleotide disulfide linkages were successfully used for covalent trapping of cysteine containing protein. In particular, an efficient conjugation of DNA methyltransferase SsoII to sequence-specific decoys was demonstrated. The obtained results assume that synthetic oligodeoxyribonucleotides bearing a new trapping site can be used as new tools to study and manipulate biological systems. [Copyright &y& Elsevier]

DNA mismatch repair (MMR) plays a crucial role in the maintenance of genomic stability. The main MMR protein, MutS, was recently shown to recognize the G-quadruplex (G4) DNA structures, which, along with regulatory functions, have a negative impact on genome integrity. Here, we studied the effect of G4 on the DNA-binding activity of MutS from Rhodobacter sphaeroides (methyl-independent MMR) in comparison with MutS from Escherichia coli (methyl-directed MMR) and evaluated the influence of a G4 on the functioning of other proteins involved in the initial steps of MMR. For this purpose, a new DNA construct was designed containing a biologically relevant intramolecular stable G4 structure flanked by double-stranded regions with the set of DNA sites required for MMR initiation. The secondary structure of this model was examined using NMR spectroscopy, chemical probing, fluorescent indicators, circular dichroism, and UV spectroscopy. The results unambiguously showed that the d(GGGT)4 motif, when embedded in a double-stranded context, adopts a G4 structure of a parallel topology. Despite strong binding affinities of MutS and MutL for a G4, the latter is not recognized by E. coli MMR as a signal for repair, but does not prevent MMR processing when a G4 and G/T mismatch are in close proximity. [ABSTRACT FROM AUTHOR]

Type II restriction–modification (RM) systems are the most widespread bacterial antiviral defence mechanisms. DNA methyltransferase SsoII (M.SsoII) from a Type II RM system SsoII regulates transcription in its own RM system in addition to the methylation function. DNA with a so-called regulatory site inhibits the M.SsoII methylation activity. Using circular permutation assay, we show that M.SsoII monomer induces DNA bending of 31° at the methylation site and 46° at the regulatory site. In the M.SsoII dimer bound to the regulatory site, both protein subunits make equal contributions to the DNA bending, and both angles are in the same plane. Fluorescence of TAMRA, 2-aminopurine, and Trp was used to monitor conformational dynamics of DNA and M.SsoII under pre-steady-state conditions by stopped-flow technique. Kinetic data indicate that M.SsoII prefers the regulatory site to the methylation site at the step of initial protein–DNA complex formation. Nevertheless, in the presence of S-adenosyl-l-methionine, the induced fit is accelerated in the M.SsoII complex with the methylation site, ensuring efficient formation of the catalytically competent complex. The presence of S-adenosyl-l-methionine and large amount of the methylation sites promote efficient DNA methylation by M.SsoII despite the inhibitory effect of the regulatory site. [ABSTRACT FROM AUTHOR]

Ribonuclease P (RNase P) is the enzyme that endonucleolytically removes 5′-precursor sequences from tRNA transcripts in all domains of life. RNase P activities are either ribonucleoprotein (RNP) or protein-only RNase P (PRORP) enzymes, raising the question about the mechanistic strategies utilized by these architecturally different enzyme classes to catalyze the same type of reaction. Here, we analyzed the kinetics and cleavage-site selection by PRORP3 from Arabidopsis thaliana ( At PRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either R p- or S p-phosphorothioate, or 2′-deoxy, 2′-fluoro, 2′-amino, or 2′-O-methyl substitutions. We observed a small but robust rescue effect of S p-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd 2 + ions, consistent with metal-ion coordination to the (pro-) S p-oxygen during catalysis. S p-phosphorothioate, 2′-deoxy, 2′-amino, and 2′- O -methyl modification redirected the cleavage mainly to the next unmodified phosphodiester in the 5′-direction. Our findings are in line with the 2′-OH substituent at nucleotide − 1 being involved in an H-bonding acceptor function. In contrast to bacterial RNase P, At PRORP3 was found to be able to utilize the canonical and upstream cleavage site with similar efficiency (corresponding to reduced cleavage fidelity), and the two cleavage pathways appear less interdependent than in the bacterial RNA-based system. [ABSTRACT FROM AUTHOR]

Abstract: Thymine glycol (Tg) in DNA is a biologically active oxidative damage caused by ionizing radiation or oxidative stress. Due to chirality of C5 and C6 atoms, Tg exists as a mixture of two pairs of cis and trans diastereomers: 5R cis–trans pair (5R,6S; 5R,6R) and 5S cis–trans pair (5S,6R; 5S,6S). Of all the modified pyrimidine lesions that have been studied to date, only thymine glycol represents a strong block to high-fidelity DNA polymerases in vitro and is lethal in vivo. Here we describe the preparation of thymine glycol-containing oligonucleotides and the influence of the oxidized residue on the structure of DNA in different sequence contexts, thymine glycol being paired with either adenine or guanine. The effect of thymine glycol on biochemical processing of DNA, such as biosynthesis, transcription and repair in vitro and in vivo, is also reviewed. Special attention is paid to stereochemistry and 5R cis–trans epimerization of Tg, and their relation to the structure of DNA double helix and enzyme-mediated DNA processing. Described here are the comparative structure and properties of other forms of pyrimidine base oxidation, as well as the role of Tg in tandem lesions. [Copyright &y& Elsevier]

The DNA mismatch repair (MMR) system plays a crucial role in the prevention of replication errors and in the correction of some oxidative damages of DNA bases. In the present work the most abundant oxidized pyrimidine lesion, 5,6-dihydro-5,6-dihydroxythymidine (thymidine glycol, Tg) was tested for being recognized and processed by the E. coli MMR system, namely complex of MutS, MutL and MutH proteins. In a partially reconstituted MMR system with MutS-MutL-MutH proteins, G/Tg and A/Tg containing plasmids failed to provoke the incision of DNA. Tg residue in the 30-mer DNA duplex destabilized double helix due to stacking disruption with neighboring bases. However, such local structural changes are not important for E. coli MMR system to recognize this lesion. A lack of repair of Tg containing DNA could be due to a failure of MutS (a first acting protein of MMR system) to interact with modified DNA in a proper way. It was shown that Tg in DNA does not affect on ATPase activity of MutS. On the other hand, MutS binding affinities to DNA containing Tg in G/Tg and A/Tg pairs are lower than to DNA with a G/T mismatch and similar to canonical DNA. Peculiarities of MutS interaction with DNA was monitored by Förster resonance energy transfer (FRET) and fluorescence anisotropy. Binding of MutS to Tg containing DNAs did not result in the formation of characteristic DNA kink. Nevertheless, MutS homodimer orientation on Tg-DNA is similar to that in the case of G/T-DNA. In contrast to G/T-DNA, neither G/Tg- nor A/Tg-DNA was able to stimulate ADP release from MutS better than canonical DNA. Thus, Tg residue in DNA is unlikely to be recognized or processed by the E. coli MMR system. Probably, the MutS transformation to active "sliding clamp" conformation on Tg-DNA is problematic.

Bacterial 6S RNAs bind to the housekeeping RNA polymerase (σ(A)-RNAP in Bacillus subtilis) to regulate transcription in a growth phase-dependent manner. B. subtilis expresses two 6S RNAs, 6S-1 and 6S-2 RNA, with different expression profiles. We show in vitro that 6S-2 RNA shares hallmark features with 6S-1 RNA: Both (1) are able to serve as templates for pRNA transcription; (2) bind with comparable affinity to σ(A)-RNAP; (3) are able to specifically inhibit transcription from DNA promoters, and (4) can form stable 6S RNA:pRNA hybrid structures that (5) abolish binding to σ(A)-RNAP. However, pRNAs of equal length dissociate faster from 6S-2 than 6S-1 RNA, owing to the higher A,U-content of 6S-2 pRNAs. This could have two mechanistic implications: (1) Short 6S-2 pRNAs (<10 nt) dissociate faster instead of being elongated to longer pRNAs, which could make it more difficult for 6S-2 RNA-stalled RNAP molecules to escape from the sequestration; and (2) relative to 6S-1 RNA, 6S-2 pRNAs of equal length will dissociate more rapidly from 6S-2 RNA after RNAP release, which could affect pRNA turnover or the kinetics of 6S-2 RNA binding to a new RNAP molecule. As 6S-2 pRNAs have not yet been detected in vivo, we considered that cellular RNAP release from 6S-2 RNA might occur via 6S-1 RNA displacing 6S-2 RNA from the enzyme, either in the absence of pRNA transcription or upon synthesis of very short 6S-2 pRNAs (∼ 5-mers, which would escape detection by deep sequencing). However, binding competition experiments argued against these possibilities.

The DNA repair protein MutS forms clamp-like structures on DNA that search for and recognize base mismatches leading to ATP-transformed signaling clamps. In this study, the mobile MutS clamps were trapped on DNA in a functional state using single-cysteine variants of MutS and thiol-modified homoduplex or heteroduplex DNA. This approach allows stabilization of various transient MutS-DNA complexes and will enable their structural and functional analysis.

A novel method for regulating the activity of homodimeric proteins--"molecular gate" approach--was proposed and its usefulness illustrated for the type II restriction endonuclease SsoII (R.SsoII) as a model. The "molecular gate" approach is based on the modification of R.SsoII with azobenzene derivatives, which allows regulating DNA binding and cleavage via illumination with light. R.SsoII variants with single cysteine residues introduced at selected positions were obtained and modified with maleimidoazobenzene derivatives. A twofold change in the enzymatic activity after illumination with light of wavelengths of 365 and 470 nm, respectively, was demonstrated when one or two molecules of azobenzene derivatives were attached to the R.SsoII at the entrance of or within the DNA-binding site.

An efficient method for the synthesis of DNA or RNA oligonucleotide 2'-hydrazides is described. Fully deprotected oligonucleotides containing a hydrazide group at the 2'-position of a uridine residue were obtained by a novel two-step procedure: periodate cleavage of an oligonucleotide with 1,2-diol group followed by conversion of the aldehyde to hydrazide with an extended linker arm using a homobifunctional reagent succinic dihydrazide and NaBH(3)CN. The resulting oligonucleotide 2'-hydrazides were efficiently conjugated by a click-type reaction at acidic pH to aliphatic or aromatic aldehydes with or without NaBH(3)CN reduction to afford novel 2'-conjugates.

We developed a metal ion chelate-assisted ligation for SNP detection by microarray. An oligonucleotide probe was separated into two 9-10-mers bearing iminodiacetic residues at the gap point. Duplex formation with the DNA target was possible only if nickel ions were added, but a nucleotide substitution opposite the gap point prevented duplex formation. Here we demonstrate the application of this approach for SNP detection (A1298C) within the 5,10-methylenetetrahydrofolate reductase gene on a microarray.