Involvement of E. coli 6S RNA in Oxidative Stress Response.

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.

Reactive Acrylamide-Modified DNA Traps for Accurate Cross-Linking with Cysteine Residues in DNA-Protein Complexes Using Mismatch Repair Protein MutS as a Model.

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.

Responses of DNA Mismatch Repair Proteins to a Stable G-Quadruplex Embedded into a DNA Duplex Structure.

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.

Kinetic Basis of the Bifunctionality of SsoII DNA Methyltransferase.

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.

Peculiarities of the interaction of the restriction endonuclease BspD6I with DNA containing its recognition site.

BACKGROUND: Nicking endonucleases are enzymes that recognize specific sites in double-stranded DNA and cleave only one strand at a predetermined position. These enzymes are involved in DNA replication and repair; they can also function as subunits of bacterial heterodimeric restriction endonucleases. One example of such a proteins is the restriction endonuclease BspD6I (R.BspD6I) from Bacillus species strain D6, which consists of the large subunit - nicking endonuclease BspD6I (Nt.BspD6I), and the small subunit (ss.BspD6I). Nt.BspD6I can function independently. Similar enzymes are now widely used in numerous biotechnological applications. The aim of this study was to investigate the fundamental properties of two subunits of R.BspD6I and their interdependence in the course of R.BspD6I activity. METHODS: The binding and hydrolysis of DNA duplexes by R.BspD6I are primary analyzed by gel electrophoresis. To elucidate the difference between Nt.BspD6I interaction with the substrate and product of hydrolysis, the thickness shear mode acoustic method is used. RESULTS AND CONCLUSIONS: The thermodynamic and kinetic parameters of the Nt.BspD6I interaction with DNA are determined. For the first time we demonstrated that Nt.BspD6I bends the DNA during complex formation. Nt.BspD6I is able to form complexes with the product nicked in the top strand and ss.BspD6I cleaves the bottom strand of the DNA consecutively. Furthermore, the influence of dA methylation in the R.BspD6I recognition site on ss.BspD6I activity is analyzed. GENERAL SIGNIFICANCE: The obtained results provide evidence that Nt.BspD6I coordinates the activity of R.BspD6I by strictly coupling of the bottom strand cleavage by ss.BspD6I to the top strand cleavage.

Mechanistic comparison of Bacillus subtilis 6S-1 and 6S-2 RNAs--commonalities and differences.

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.

Thermo-switchable activity of the restriction endonuclease SsoII achieved by site-directed enzyme modification.

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 °Ð¡ that almost abolished the enzymatic activity when compared with the unmodified enzyme. However, heating of the modified enzyme to 45 °Ð¡ 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.

Restriction endonuclease SsoII with photoregulated activity--a "molecular gate" approach.

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.

Kinetic Analysis of the Interaction of Nicking Endonuclease BspD6I with DNA.

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.

DNA polymerases beta and lambda bypass thymine glycol in gapped DNA structures.

Here we investigated the ability of the human X-family DNA polymerases beta and lambda to bypass thymine glycol (Tg) in gapped DNA substrates with the damage located in a defined position of the template strand. Maximum velocities and the Michaelis constant values were determined to study DNA synthesis in the presence of either Mg(2+) or Mn(2+). Additionally, the influence of hRPA (human replication protein A) and hPCNA (human proliferating cell nuclear antigen) on TLS (translesion synthesis) activity of DNA polymerases beta and lambda was examined. The results show that (i) DNA polymerase lambda is able to catalyze DNA synthesis across Tg, (ii) the ability of DNA polymerase lambda to elongate from a base paired to a Tg lesion is influenced by the size of the DNA gap, (iii) hPCNA increases the fidelity of Tg bypass and does not influence normal DNA synthesis catalyzed by DNA polymerase lambda, (iv) DNA polymerase beta catalyzes the incorporation of all four dNTPs opposite Tg, and (v) hPCNA as well as hRPA has no specific effect on TLS in comparison with the normal DNA synthesis catalyzed by DNA polymerase beta. These results considerably extend our knowledge concerning the ability of specialized DNA polymerases to cope with a very common DNA lesion such as Tg.

Similarities and differences between 6S RNAs from Bradyrhizobium japonicum and Sinorhizobium meliloti.

6S RNA, a conserved and abundant small non-coding RNA found in most bacteria, regulates gene expression by inhibiting RNA polymerase (RNAP) holoenzyme. 6S RNAs from α-proteobacteria have been studied poorly so far. Here, we present a first in-depth analysis of 6S RNAs from two α-proteobacteria species, Bradyrhizobium japonicum and Sinorhizobium meliloti. Although both belong to the order Rhizobiales and are typical nitrogen-fixing symbionts of legumes, their 6S RNA expression profiles were found to differ: B. japonicum 6S RNA accumulated in the stationary phase, thus being reminiscent of Escherichia coli 6S RNA, whereas S. meliloti 6S RNA level peaked at the transition to the stationary phase, similarly to Rhodobacter sphaeroides 6S RNA. We demonstrated in vitro that both RNAs have hallmarks of 6S RNAs: they bind to the σ70-type RNAP holoenzyme and serve as templates for de novo transcription of so-called product RNAs (pRNAs) ranging in length from ∼13 to 24 nucleotides, with further evidence of the synthesis of even longer pRNAs. Likewise, stably bound pRNAs were found to rearrange the 6S RNA structure to induce its dissociation from RNAP. Compared with B. japonicum 6S RNA, considerable conformational heterogeneity was observed for S. meliloti 6S RNA and its complexes with pRNAs, even though the two 6S RNAs share ∼75% sequence identity. Overall, our findings suggest that the two rhizobial 6S RNAs have diverged with respect to their regulatory impact on gene expression throughout the bacterial life cycle.

Metal ion CHElate-aSSisted LIGAtion (CHESS LIGA) for SNP detection on microarrays.

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.

A study on endonuclease BspD6I and its stimulus-responsive switching by modified oligonucleotides.

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.

Preparation of 2'-hydrazino oligonucleotides and their reaction with aldehydes and 1,3-diketones.

Oligodeoxyribonucleotides that contain a hydrazino nucleoside, 2'-O-(2-hydrazinoethyl)uridine were prepared and shown to react with aldehydes or 1,3-diketones with the formation of hydrazones or pyrazoles, respectively. The method may be applicable for the preparation of oligonucleotide-peptide conjugates.

A convenient solid-phase method for the synthesis of novel oligonucleotide-folate conjugates.

We describe the preparation of two batches of a polymer support for the incorporation of folic acid into oligonucleotides. The method permits the regioselective attachment of a target nucleic acid sequence through its 3'-end to either the alpha-or gamma-carboxyl group of L-glutamic acid, respectively. The supports have been tested in solid-phase synthesis of oligonucleotide-folate conjugates for cell delivery studies.

Adsorption of synthetic homo- and hetero-oligodeoxynucleotides onto highly oriented pyrolytic graphite: atomic force microscopy characterization.

DNA adsorption on electrode surfaces is of fundamental interest for the development of DNA-based biosensors. The free adsorption of 10-mer synthetic oligodeoxynucleotides (ODNs) onto highly oriented pyrolytic graphite (HOPG) surfaces was studied using Magnetic AC mode atomic force microscopy (MAC Mode AFM). The mechanism of interaction of nucleic acids with carbon electrode surfaces was elucidated, using 10-mer synthetic homo- and hetero-ODNs sequences of known base sequences, because they allow clear interpretation of the experimental data. AFM images in air revealed different adsorption patterns and degree of HOPG surface coverage for the ODNs, and correlation with the individual structure and base sequence of each ODN molecule will be presented. The results demonstrated that the hydrophobic interactions with the HOPG hydrophobic surface explain the main adsorption mechanism, although other effects such as electrostatic and Van der Waals interactions may contribute to the free adsorption process. The ODNs interacted differently with the HOPG surface, according to the ODN sequence hydrophobic characteristics, being directly depending on the molecular mass, the hydrophobic character of the individual bases and on the secondary structure of the molecule. The importance of the type of base existent at the ODN chain extremities on the adsorption process was investigated and different adsorption patterns were obtained with ODN sequences composed by the same group of bases aligned in a different order.

Analysis of the Cleavage Mechanism by Protein-Only RNase P Using Precursor tRNA Substrates with Modifications at the Cleavage Site.

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 (AtPRORP3) using precursor tRNAs (pre-tRNAs) with individual modifications at the canonical cleavage site, with either Rp- or Sp-phosphorothioate, or 2'-deoxy, 2'-fluoro, 2'-amino, or 2'-O-methyl substitutions. We observed a small but robust rescue effect of Sp-phosphorothioate-modified pre-tRNA in the presence of thiophilic Cd2+ ions, consistent with metal-ion coordination to the (pro-)Sp-oxygen during catalysis. Sp-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, AtPRORP3 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.

Specific conjugation of DNA binding proteins to DNA templates through thiol-disulfide exchange.

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.

Synthesis and applications of oligonucleotide-carbohydrate conjugates.

Nowadays, oligonucleotide-carbohydrate conjugates are used in antisense biotechnology and in the study of glycosylated DNA functioning in vitro. The application of mono- and disaccharide phosphoramidites, solid-phase supports with immobilized carbohydrates, glycosylated nucleoside phosphoramidites, and postsynthetic conjugation of reactive sugar derivatives with oligonucleotides for preparation of oligonucleotide-carbohydrate conjugates have been systematically studied. The advantages and disadvantages of these approaches are considered. Possible strategies for synthesis of glycoclusters with different topologies conjugated to DNA are discussed. Applications of oligonucleotide-carbohydrate conjugates are highlighted. Studies of interactions of glycosylated oligonucleotides with proteins and effective cell-specific delivery of oligonucleotide-carbohydrate conjugates are discussed.

Synthesis of (2'S)- and (2'R)-2'-deoxy-2'-[(2-methoxyethoxy)amino] pyrimidine nucleosides and oligonucleotides.

Syntheses of specified 2'-modified nucleosides were achieved: a) via oximation of the 5',3'-blocked 2'-oxocytidine, followed by reduction, or b) by intramolecular nucleophilic addition of 3'-(2-methoxyethoxy)carbamate to the 2'-position with opening of O(2),2'-anhydrouridine. For the first time, 3'-phosphoroamidites of these 2'-modified nucleosides were successfully incorporated into oligonucleotides by solid-phase synthesis. Incorporation of 2'-modified nucleotides into oligodeoxyribonucleotides had a negative effect on the duplex T(m) values with the DNA or RNA complements. Nevertheless, modified nucleotides have shown good target recognition; the (S)-isomer binds preferably to RNA and the (R)-isomer to DNA. Both modified nucleosides significantly increased nuclease resistance of the oligodeoxyribonucleotides.