[MutL Protein from the Neisseria gonorrhoeae Mismatch Repair System: Interaction with ATP and DNA].

The mismatch repair system (MMR) ensures the stability of genetic information during DNA replication in almost all organisms. Mismatch repair is initiated after recognition of a non-canonical nucleotide pair by the MutS protein and the formation of a complex between MutS and MutL. Eukaryotic and most bacterial MutL homologs function as endonucleases that introduce a single-strand break in the daughter strand of the DNA, thus activating the repair process. However, many aspects of the functioning of this protein remain unknown. We studied the ATPase and DNA binding functions of the MutL protein from the pathogenic bacterium Neisseria gonorrhoeae (NgoMutL), which exhibits endonuclease activity. For the first time, the kinetic parameters of ATP hydrolysis by the full-length NgoMutL protein were determined. Its interactions with single- and double-stranded DNA fragments of various lengths were studied. NgoMutL was shown to be able to efficiently form complexes with DNA fragments that are longer than 40 nucleotides. Using modified DNA duplexes harboring a 2-pyridyldisulfide group on linkers of various lengths, we obtained NgoMutL conjugates with DNA for the first time. According to these results, the Cys residues of the wild-type protein are located at a distance of approximately 18-50 Šfrom the duplex. The efficiency of the affinity modification of Cys residues in NgoMutL with reactive DNAs was shown to decrease in the presence of ATP or its non-hydrolyzable analog, as well as ZnCl2, in the reaction mixture. We hypothesize that the conserved Cys residues of the C-terminal domain of NgoMutL, which are responsible for the coordination of metal ions in the active center of the protein, are involved in its interaction with DNA. This information may be useful in reconstruction of the main stages of MMR in prokaryotes that are different from γ-proteobacteria, as well as in the search for new targets for drugs against N. gonorrhoeae.

[The Role of Cysteine Residues in the Interaction of Nicking Endonuclease BspD6I with DNA].

Nicking endonucleases (NEs) are a small, poorly studied family of restriction endonucleases. The enzymes recognize a target sequence in DNA, but catalyze the hydrolysis of only one strand. The mechanism of their action is important to study because NEs with new specificities are necessary to design to solve the practical tasks of biotechnology. One of the modern approaches for investigation of protein-nucleic acid interactions is fluorescence spectroscopy, which involves the introduction of fluorophores into proteins, mainly through Cys residues due to the high reactivity of their thiol group. To implement this approach, it is necessary to clarify the role of Cys residues in the functioning of the native protein and the possible consequences of their modification. Crosslinking was used to study whether Cys residues are close to DNA in the complex with NE BspD6I. Reactions were carried out using the wild-type enzyme, its mutant form NE BspD6I(C11S/C160S), and modified DNA duplexes containing the 2-pyridyldisulfide group at the C2' atom of the sugar-phosphate moiety in different positions of the oligonucleotide strand. The Cys residues of NE BspD6I were for the first time shown to be in close proximity to DNA during the binding process, including the step of a nonspecific complex formation. The substitutions C11S and C160S in the N-terminal domain of the enzyme slightly decreased the efficiency of substrate hydrolysis. Construction of a cysteine-free NE BspD6I variant and examination of its properties will provide additional information about the functional significance of the Cys residues for this unique enzyme.

Endonuclease Activity of MutL Protein of the Rhodobacter sphaeroides Mismatch Repair System.

We have purified the MutL protein from Rhodobacter sphaeroides mismatch repair system (rsMutL) for the first time. rsMutL demonstrated endonuclease activity in vitro, as predicted by bioinformatics analysis. Based on the alignment of 1483 sequences of bacterial MutL homologs with presumed endonuclease activity, conserved functional motifs and amino acid residues in the rsMutL sequence were identified: five motifs comprising the catalytic site responsible for DNA cleavage were found in the C-terminal domain; seven conserved motifs involved in ATP binding and hydrolysis and specific to the GHKL family of ATPases were found in the N-terminal domain. rsMutL demonstrated the highest activity in the presence of Mn2+. The extent of plasmid DNA hydrolysis declined in the row Mn2+ > Co2+ > Mg2+ > Cd2+; Ni2+ and Ca2+ did not activate rsMutL. Divalent zinc ions inhibited rsMutL endonuclease activity in the presence of Mn2+ excess. ATP also suppressed plasmid DNA hydrolysis by rsMutL. Analysis of amino acid sequences and biochemical properties of five studied bacterial MutL homologs with endonuclease activity revealed that rsMutL resembles the MutL proteins from Neisseria gonorrhoeae and Pseudomonas aeruginosa.

Small Noncoding 6S RNAs of Bacteria.

Small noncoding RNAs (ncRNAs) are non-translated transcripts with lengths below 300 nucleotide residues. Regulation of cellular processes under the influence of these ncRNAs is the most various in eukaryotic cells, but numerous ncRNAs are also found in bacteria. One of the best-known small prokaryotic ncRNAs is 6S RNA - it has been detected in all branches of bacteria. Due to their conserved secondary structure including a large central "loop" flanked by long double-helical arms, 6S RNAs can bind holoenzymes of RNA polymerase (RNAP) and inhibit their activity. This inhibits transcription of many genes. According to data of comparative transcriptome analysis, the 6S RNA-dependent regulation of transcription affects the expression level of hundreds of genes involved in various cellular processes. 6S RNA has the unique feature of serving as a transcription template for the synthesis of short product RNAs (pRNAs) complementary to the central part of the molecule. The length and abundance of pRNAs vary depending on the physiological status of the cell. The synthesis of pRNAs is of great importance because it releases RNAP and provides reversibility of the inhibition. A similar mechanism has been described for the noncoding mouse B2 RNA that inhibits the activity of RNAP II. This finding can be taken as evidence for the common evolutionary origin of the ncRNA-dependent regulation of RNAP and its immense significance for cells. This review summarizes the state of knowledge about the main features and functions of 6S RNAs from various bacterial species with a special focus on the peculiarities of pRNA synthesis. The majority of functional insights on 6S RNAs have been gained for E. coli 6S RNA as the best-studied model system.

Regulation of activity of transcription factor NF-κB by synthetic oligonucleotides.

Eukaryotic dimeric nuclear factor-κB (NF-κB) is one of the main transcription factors that activate expression of genes, products of which play the key role in development of cardiovascular pathologies, carcinogenesis, and inflammatory and viral diseases. In this review, the main attention is given to modulation of the transcription factor NF-κB activity by antisense oligonucleotides and oligonucleotide decoys. Also, current concepts about interactions between NF-κB dimers and DNA and general problems that arise in experimental use of synthetic oligonucleotides in vivo are discussed.

[2'-Modified oligoribonucleotides, containing 1,2-diol and aldehyde groups. Synthesis and properties].

1,2-Diol-oligoribonucleotides were prepared using fully protected 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite. Incorporation of the 2'-modified uridine residue into oligonucleotide chains does not significantly affect the thermal stability of RNA and RNA-DNA duplexes. Periodate oxidation of the 1,2-diol results in reactive 2'-aldehyde oligoribonucleotides. Further application of these oligonucleotides for cross-linking with bacterial ribonuclease P was investigated.

[Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations].

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

The study of the interaction of (cytosine-5)-DNA methyltransferase SsoII with DNA by acoustic method.

The interaction of (cytosine-5)-DNA methyltransferase SsoII (M.SsoII) with double-stranded DNA was studied by means of thickness shear mode acoustic method (TSM) and gel electrophoresis. M.SsoII recognizes in double-stranded DNA the methylation site 5'-CCNGG-3' (N=A, C, G, T) and methylates the inner cytosine residue. M.SsoII also acts as a transcription factor via binding to the regulatory site 5'-AGGACAAATTGTCCT-3' in the promoter region of SsoII restriction-modification system. We designed three 60-mer biotinylated DNA duplexes: with the methylation site (60met), with the regulatory site (60reg), and without a specific binding site (60oct). A strong binding of M.SsoII with each one of the studied DNA immobilized on the TSM transducer has been shown. The equilibrium dissociation constants, K(D), of the M.SsoII-DNA complexes decreased in the order 60oct>60reg>60met, suggesting a higher stability of M.SsoII-60met complex in comparison with the others. The association rate constant, k(a), was also higher for 60met, while similar values were obtained for 60reg and 60oct. The difference in the kinetic parameters for 60met and 60reg suggested a possible way of coordination between the two M.SsoII functions in a cell.

[Secondary structure of SsoII-like (cytosine-5)-DNA methyltransferases N-terminal region determined by circular dichroism spectroscopy].

(Cytosine-5)-DNA methyltransferase SsoII (M.SsoII) has a long N-terminal region (1-71 residues) preceding the sequence with conservative motifs, which are characteristic for all DNA methyltransferases of such kind. The presence of this region provides M.SsoII capability to act as a transcription regulator in SsoII restriction-modification system. To perform its regulatory function, M.SsoII binds specifically to a 15-mer inverted repeat in the promoter region of SsoII restriction-modification system genes. In the present work, properties of the protein delta(72-379)M.Ecl18kI are studied, which is a deletion mutant of the SsoII-like DNA-methyltransferase M.Ecl18kI and is homologous to M.SsoII N-terminal region. delta(72-379)M.Ecl18kI capability to bind specifically a DNA duplex containing the regulatory site is demonstrated. However, such a binding takes place only in the presence of high protein excess relative to DNA, which could indicate an altered structure in the deletion mutant in comparison with the full-length M.SsoII. Circular dichroism spectroscopy demonstrated that delta(72-379)M.Ecl18kI has a strongly pronounced secondary structure and contains 32% a-helices and 20% beta-sheets. Amino acid sequences alignment of M.SsoII N-terminal region and transcription factors of known spatial structure is made. An assumption is made how alpha-helices and beta-sheets are arranged in M.SsoII N-terminal region.

[2'-aldehyde oligonucleotides: synthesis and use for affinity modification of DNA-recognizing proteins].

Oligonucleotides with 1,2-diol grouping were prepared from 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxo-ethyl]uridine 3'-phosphoramidite. The thermal stability of modified DNA duplexes and their ability to form complexes with the p50 subunit of the NF-kappaB transcription factor and (cytosine-5)-DNA methyltransferase SsoII were studied. The periodate oxidation of the l,2-diol grouping of the oligonucleotides resulted in reactive 2'-aldehyde derivatives. The opportunity of their use for the affinity modification of DNA-recognizing proteins was studied.

SsoII-like DNA-methyltransferase Ecl18kI: interaction between regulatory and methylating functions.

The interaction of DNA-methyltransferase Ecl18kI (M.Ecl18kI) with a fragment of promoter region of restriction-modification system SsoII was studied. It is shown that dissociation constants of M.Ecl18kI and M.SsoII complexes with DNA ligand carrying a regulatory site previously characterized for M.SsoII have comparable values. A deletion derivative of M.Ecl18kI, Delta(72-379)Ecl18kI, representing the N-terminal protein region responsible for regulation, was obtained. It is shown that such polypeptide fragment has virtually no interaction with the regulatory site. Therefore, the existence of a region responsible for methylation is necessary for maintaining M.Ecl18kI regulatory function. The properties of methyltransferase NlaX, which is actually a natural deletion derivative of M.Ecl18kI and M.SsoII lacking the first 70 amino acid residues and not being able to regulate gene expression of the SsoII restriction-modification system, were studied. The ability of mutant forms of M.Ecl18kI incorporating single substitutions in regions responsible for regulation and methylation to interact with both sites of DNA recognition was characterized. The data show a correlation between DNA-binding activity of two M.Ecl18kI regions-regulatory and methylating.

[New derivatives of azobenzene for the directed modification of proteins].

Derivatives of azobenzene which contained a maleimide group in one of the benzene rings (for binding to a protein cysteine residue) and maleimide, hydroxyl, or carboxyl substitutes in another benzene ring were synthesized. The reactivity of these compounds towards a cysteine residue of a protein and their optical properties in a free state and after their attachment to the mutant forms of the SsoII restriction endonuclease were studied.

[Synthesis and characteristics of modified DNA fragments containing thymidine glycol residues].

Chemical synthesis of a series of modified oligodeoxyribonucleotides containing one or two residues of thymidine glycol (5,6-dihydro-5,6-dihydroxythymidine), the main product of oxidative DNA damage, is described. The thermal stability of DNA duplexes containing thymidine glycol residues was studied using UV spectroscopy. Introduction of even one thymidine glycol residue into the duplex structure was shown to result in its significant destabilization. Data on the interaction of DNA methyltransferases and type II restriction endonucleases with DNA ligands containing oxidized thymine were obtained for the first time. Introduction of a thymidine glycol residue into the central degenerate position of the recognition site of restriction endonuclease SsoII was found to result in an increase in the initial hydrolysis rate of the modified duplex in comparison with that of the unmodified structure. The affinity of C5-cytosine methyltransferase SsoII for the DNA duplex bearing thymidine glycol was found to be twofold higher than for the unmodified substrate. However, such a modification of the DNA ligand prevents its methylation. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

Non-Coding RNAs As Transcriptional Regulators In Eukaryotes.

Non-coding RNAs up to 1,000 nucleotides in length are widespread in eukaryotes and fulfil various regulatory functions, in particular during chromatin remodeling and cell proliferation. These RNAs are not translated into proteins: thus, they are non-coding RNAs (ncRNAs). The present review describes the eukaryotic ncRNAs involved in transcription regulation, first and foremost, targeting RNA polymerase II (RNAP II) and/or its major proteinaceous transcription factors. The current state of knowledge concerning the regulatory functions of SRA and TAR RNA, 7SK and U1 snRNA, GAS5 and DHFR RNA is summarized herein. Special attention is given to murine B1 and B2 RNAs and human Alu RNA, due to their ability to bind the active site of RNAP II. Discovery of bacterial analogs of the eukaryotic small ncRNAs involved in transcription regulation, such as 6S RNAs, suggests that they possess a common evolutionary origin.

Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking.

A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG). For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was approximately 50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of Sso II to its DNA substrate.

[Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering].

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.

Oligonucleotide cleavage by restriction endonucleases MvaI and EcoRII: a comprehensive study on the influence of structural parameters on the enzyme-substrate interaction.

To elucidate the mechanism of action of the restriction endonucleases--isoschizomers EcoRII and MvaI--a study was made of their interaction with a set of synthetic oligonucleotide duplexes containing a single 5'-d(CCA/TGG)-3' EcoRII (MvaI) recognition site. The substrates had varying length and structure of the nucleotide sequences flanking the recognition site. The structure of the flanking sequence is important for the cleavage by EcoRII and MvaI enzymes; there is a structure which was found to speed up the EcoRII and MvaI action. The cleavage of oligonucleotide duplexes by EcoRII enzyme does not go to completion. EcoRII endonuclease cleaved extended substrates less efficiently than short ones. Extension of the flanking sequences, with the same nucleotide surrounding of the recognition site, substantially altered the whole kinetic pattern of MvaI hydrolysis. This was not observed with EcoRII enzyme. The restriction endonuclease MvaI distinguished between dA and dT residues in the recognition site, which was reflected in the higher rate of hydrolysis of the dA-containing strand of the quasi-palindromic DNA duplex.

Chemical cross-linking of MvaI and EcoRII enzymes to DNA duplexes containing monosubstituted pyrophosphate internucleotide bond.

DNA duplexes containing a monosubstituted pyrophosphate internucleotide group, instead of a phosphodiester bond, were used as cross-linking reagent for the affinity modification of the restriction endonucleases EcoRII and MvaI (R.EcoRII and R.MvaI). An active group was introduced into the enzyme's recognition site or between the recognition site and flanking sequence. The substrate properties of such DNA duplexes were determined. Cross-linking specificity was demonstrated by competition experiments with unmodified substrate, as well as by the absence of cross-linking to an active duplex lacking a recognition site. It was shown that the nucleophilicity of the buffer solution and the presence of the enzyme cofactor Mg2+ dramatically affected the cross-linking yield.

Interaction of the MvaI and SsoII methyltransferases with DNAs altered at the central base pair of the recognition sequence.

The interaction of the MvaI and SsoII DNA methyltransferases (MTases; M.MVaI and M.SsoII, respectively) with a set of synthetic DNA duplexes, containing a M.MvaI and M.SsoII recognition site (CCWGG), was investigated. In these DNA duplexes dA or dT of the recognition site was replaced by nucleoside analogs with modified sugar moieties and heterocyclic bases (2'-deoxy-2'-fluorouridine (flU), 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (xT), 1-(beta-D-3'-deoxy-threo-pentofuranosyl)uracil (tU)), or by 1,3-propanediol (Prd). A new approach for monitoring methylation of each strand of DNA duplexes by MTases was developed. It allowed the determination of the influence of the modification in one DNA strand on the methylation of the other. In most cases, for both M.MvaI and M.SsoII, sugar analog-containing duplexes showed inhibition of methylation of only the modified strand. Prd-containing DNA duplexes were not substrates for M.MvaI. M.SsoII did not methylate DNA duplexes in which the dT residue was replaced by Prd.

Modified substrates as probes for studying uracil-DNA glycosylase.

In order to study the mechanism of action of uracil-DNA glycosylase (UDG) from human placenta, single-stranded (ss) and double-stranded (ds) oligodeoxyribonucleotides (oligos), containing deoxyuridine (dU) and a wide variety of their analogs were used. It was shown that UDG has a twofold preference for ss oligos over ds oligos and a twofold preference for intermolecular duplexes over similar hairpin-like duplexes. The replacement of dU with 1-(beta-D-2'-deoxy-threo-pentofuranosil)uracil (xU) or 1-(beta-D-3'-deoxy-threo-pentofuranosil)uracil (tU), which results in a change in sugar hydroxyl configuration, has no influence on UDG binding to such substrates, but inhibits uracil removal. A oligo containing 2'-deoxy-2'-fluorouridine (flU), with a 3'-endo conformation of modified sugar is recognized by UDG 100-200-fold less efficiently than the natural ones. F or Br atoms or a methyl group were introduced at position 5 of a dU residue in an oligo. It was shown that the nature of a substituent at this position is essential for UDG function.