[The structure and mechanism of the action of type-IB DNA topoisomerases].

DNA topoisomerases responsible for the superspiralization of genomic DNA participate in almost all vitally important cell processes, including replication, transcription, and recombination, and are essential for normal cell functioning. The present review summarizes published data for type-IB topoisomerases. The results concerning the thermodynamic, structural, and kinetic aspects of the functioning of topoisomerases and the peculiarities of the mechanisms of their action have been analyzed for the first time.

Structure and mechanism of action of type IA DNA topoisomerases.

DNA topoisomerases are enzymes responsible for regulation of genomic DNA supercoiling. They participate in essential processes of cells such as replication, transcription, recombination, repair, etc., and they are necessary for normal functioning of the cells. Topoisomerases alter the topological state of DNA by either passing one strand of the helix through the other strand (type I) or by passing a region of duplex DNA through another region of duplex DNA (type II). Type I DNA topoisomerases are subdivided into enzymes that bind to the 5'- (type IA) or 3'-phosphate group (type IB) during relaxation of the cleavable DNA. This review summarizes the literature on type IA DNA topoisomerases. Special attention is given to particular properties of their structure and mechanisms of functioning of these enzymes.

[Interaction of single-stranded DNA with the second DNA-binding site of RecA nucleoprotein filament].

RecA protein first forms filament on single-stranded (ss) DNA forming the first DNA-binding site for interaction with this ssDNA a formation of the second site for interaction with double-stranded DNA occurs in parallel. Then the formed nucleoprotein filament interacts with molecules of double-stranded (ds) DNA but can also recognize ssDNA. The formed complex realizes a search of homology and exchange of homologous strands. We have studied recently the mechanism of RecA filamentation on ssDNA. Here a study of interaction of different DNAs with the second site of RecA filament using a method of stepwise increase of the ligand complicity was performed. The second site under recognition interacts with every nucleotide units of DNA-ligand forming contact with both internucleotide phosphate groups and bases of DNA. Pyrimidinic d(pC)n [Russian character: see text d(pT)n oligonucleotides interact with the second site of the RecA filament more effectively than with d(pA)n oligonucleotides. This occurs due to a more effective interaction of the RecA filament with 5'-terminal unit of pyrimidinic DNAs and to a difference in specific conformational changes of nucleoprotein filaments in the complex with purinic and pyrimidinic DNAs. A comparison of thermodynamic characteristics of DNA recognition by the first and the second sites of DNA recognition is carried out. It was shown that at n >10 d(pC)n d(pN)n interact with the second site weaker, that with the first site. The complexation of the second site with d(pA)n at n >20 is more effective than with the first site. The difference in the affinity of d(pA)n to the fist and second sites is increased monotonically with the enhancement of their length. Possible mechanisms of RecA-dependent search of homology and strand exchange are discussed.

[Physico-chemical basis of RecA nucleo-protein filament formation on single].

The analysis of RecA protein playing a central role in homologous recombination of E. coli with single-stranded DNAs of various structure and length on quantitative level is carried out for the first time. It was shown that weak additive interactions between protein monomers of filament and different structural elements of DNA provide DNA recognition. Orthophosphate and dNMPs (I50 = 12-20 mM) were shown to be the minimal inhibitors of RecA filamentation on d(pN)20. The lengthening of homooligonucleotides from d(pN)2 to d(pN)20 by one unit leads to monotonic increase in the affinity by a factor approximately 2 (factor f) due to weak additive contacts of RecA with every internucleoside phosphate group of DNA (f = 1.56) and specific interactions with each of T and C bases (f = 1.32). RecA filament does not practically interact with bases of d(pA)n, but contacts with internucleoside phosphate groups of the first turn (n < 10; f = 2.1) more effective than with additional turns of d(pA)n (n > 10; f = 1.3). The affinity of RecA protein for d(pN)n, containing typical and a number of different modified bases depends on a type of base, peculiarities of DNA structure and conformation of its sugar-phosphate backbone. The affinity is increased significantly if the bases contain exocyclic proton accepting groups. The possible reasons of preferable complexation of RecA with DNA of definite structure and length are analyzed. The mechanism of single-stranded DNA recognition by RecA and hypothetical mechanism of homological DNA strands exchange are proposed.

[The mechanism of supercoiled DNA recognition by eukaryotic type I topoisomerases. II. A comparison of the enzyme interaction with specific and nonspecific oligonucleotides]. / Mekhanizm uznavaniia superskruchennoi DNK éukarioticheskimi topoizomerazami pervogo tipa II. Sravnenie vzaimodeistviia fermentov so spetsificheskimi i nespetsificheskimi oligonukleotidami.

Data on the interaction of DNA type I topoisomerases from the murine and human placenta cells with specific and nonspecific oligonucleotides of various structures and lengths are summarized. The relative contributions of various contacts between the enzymes and DNA that have previously been detected by X-ray analysis to the total affinity of the topoisomerases for DNA substrates are estimated. Factors that determine the differences in the enzyme interactions with specific and nonspecific single- and double-stranded DNAs are revealed. The results of the X-ray analysis of human DNA topoisomerase I are interpreted taking into account data on the comprehensive thermodynamic and kinetic analysis of the enzyme interaction with the specific and nonspecific DNAs.

[The mechanism of specific cleavage of supercoiled DNA by human DNA topoisomerase I: the effect of ligand structure on the catalytic step of reaction]. / Mekhanizm spetsificheskogo rasshchepleniia superskruchennoi DNK DNK-topoizomerazoi I cheloveka: vliianie struktury liganda na kataliticheskuiu stadiiu reaktsii.

Eukaryotic DNA topoisomerase I (Topo) regulates the topological state of cell DNA and plays an important part in replication, transcription, repair, and recombination. Factors affecting the specific recognition of topologically stressed DNA were analyzed on the basis of the thermodynamic and kinetic data on the Topo-DNA interaction and the X-ray data on human Topo. A model was advanced for possible structural changes occurring in the ligand after initial recognition. The effect of conformational changes in specific DNA on the catalytic stage of the reaction was analyzed.

[The mechanism of the supercoiled DNA recognition by eukaryotic type I topoisomerases. I. The enzyme interaction with nonspecific oligonucleotides]. / Mekhanizm uznavaniia superskruchennoi DNK éukarioticheskimi topoizomerazami pervogo tipa. I. Vzaimodeistvie fermentov s nespetsificheskimi oligonukleotidami.

Interaction of the DNA type I topoisomerases from the murine and human placenta cells with nonspecific oligonucleotides was analyzed. The contributions of strong and week nonspecific electrostatic, van der Waals's, and hydrophobic interactions, and hydrogen bonding of the enzymes to the complex formation with the single- and double-stranded DNAs were determined. The factors that determine the top-priority recognition of the topologically stressed DNA were revealed. The results were interpreted in comparison with the X-ray analysis data for human DNA topoisomerase I.

[Polymethylene derivatives of nucleic bases with omega-functional groups. II. Adenine and hypoxanthine derivatives]. / Polimetilenovye proizvodnye nukleinovykh osnovanii s omega-funktsional'nymi gruppami. II. Proizvodnye adenina i gipoksantina.

N9-Polymethylene derivatives of adenine and hypoxanthine with various functional groups in the omega-position of the alkyl substituent were synthesized. Their physico-chemical properties and effect on the HIV reverse transcriptase and DNA topoisomerase I were studied.

Inhibition of human DNA topoisomerase I by new DNA minor groove ligands: derivatives of oligo-1,3-thiazolecarboxamides.

A series of novel thiazole-containing oligopeptides (oligo-1,3-thiazolecarboxamides) interesting specifically with the minor groove of DNA was shown to inhibit human DNA topoisomerase I (topo I). Inhibitory effects of thiazole-containing oligopeptides (TCO) increase with the number of thiazole units in such compounds. Inhibitory properties of TCO containing 3 or 4 thiazole units were shown to be 3-10 times better than those of the well-known natural antibiotic, distamycin A containing pyrrole rings. The structure of various additional groups attached to the N-terminus and C-terminus of TCO had no significant effect on TCO interaction with the complex of DNA and topo I. TCO were shown to be capable of binding with double-stranded DNA (dsDNA), and the majority of TCO analyzed were more effective in binding with dsDNA than distamycin A. Possible reasons for the different effects of distamycin A and TCO on the reaction of relaxation catalyzed by topo I are discussed.

Formation of the D-loop structure complexes between DNA and oligonucleotides and affinity modification of DNA by chemically reactive derivatives of oligonucleotides lead to the recombination.

INTRODUCTION: Affinity modification of DNA by chemically reactive derivatives of complementary oligonucleotides (ODNs) and antisense ODNs has shown an application for the inhibition of gene expression and the growth of viruses and parasites in high organisms. Unfortunately, the rapid advancement of antisense therapeutic approaches is not parallel to the investigation of possible consequences of antisense and gene-directed ODNs on genetic material of the cells being treated. Here we tried for the first time to estimate a possible genetic impact of antisense ODNs and their chemically reactive derivatives on the cells using bacteria and the plasmid DNA. MATERIAL AND METHODS: Recombination of direct repeats, induced by the formation of reversible complexes of plasmid DNA with complementary ODNs and after covalent binding of the alkylating derivative of the ODNs with DNA, has been investigated. For this purpose, a polylinker sequence flanked by 165 bp direct repeats was inserted within the tet gene of pBR 327. This plasmid was used to construct DNA containing AT- and GC-rich sequences placed in the central region of the polylinker. RESULTS: Transformation of E. coli cells with the plasmids (and with mixtures of the plasmids with d(pN)17 complementary to the AT- and GC-rich sequences) did not produce deletions. After modification of plasmids with alkylating derivatives of d(pN)17, the deletion of the polylinker DNA region (recombination) revealed the restoration of the tet gene function. The same effect was found at the cell transformations with the D-loop complex of the plasmids with ODNs, but the frequency of the transformants was about 1.5-2 times lower. The data obtained demonstrate that the complexes of DNA with complementary ODNs and the modification of the plasmids by reactive ODN derivatives result in induction of the recombination process and in loss of genetic material.

Possibilities of the method of step-by-step complication of ligand structure in studies of protein--nucleic acid interactions: mechanisms of functioning of some replication, repair, topoisomerization, and restriction enzymes.

X-Ray structure analysis is one of the most informative methods for investigation of enzymes. However, it does not provide quantitative estimation of the relative efficiency of formation of contacts revealed by this method, and when interpreting the data this does not allow taking into account the relative contribution of some specific and nonspecific interactions to the total affinity of nucleic acids (NA) to enzymes. This often results in unjustified overestimation of the role of specific enzyme--NA contacts in affinity and specificity of enzyme action. In recent years we have developed new approaches to analysis of the mechanisms of protein--nucleic acid interactions allowing quantitative estimation of the relative contribution of virtually every nucleotide unit (including individual structural elements) to the total affinity of enzymes to long DNA and RNA molecules. It is shown that the interaction between enzymes and NA on the molecular level can be successfully analyzed by the methods of synthesis and analysis, that is, step-by-step simplification or complication of the structure of a long NA-ligand. This approach allows the demonstration that complex formation including formation of contacts between enzymes and specific NA units can provide neither high affinity of the enzymes to NA nor the specificity of their action. Using a number of sequence-independent replication and repair enzymes specifically recognizing a modified unit in DNA and also some sequence-dependent topoisomerization and restriction enzymes as examples, it was shown that virtually all nucleotide units within the DNA binding cleft interact with the enzyme, and high affinity mainly (up to 5-7 of 7-10 orders of magnitude) is provided by many weak additive interactions between these enzymes and various structural elements of the individual NA nucleotide units. At the same time, the relative contribution of specific interactions to the total affinity of NA is rather small and does not exceed 1-2 orders of magnitude. Specificity of enzyme action is provided by the stages of the enzyme-dependent NA adaptation to the optimal conformation and directly of catalysis: kcat increases by 3-7 orders of magnitude when changing from nonspecific to specific NA. In the present work we summarized our experience in studies of enzymes by the method of step-by-step complication of the ligand structure and performed a detailed analysis of the features of this approach and its possibilities for the study of protein--nucleic acid interactions on the molecular level.

Interaction of human DNA topoisomerase I with specific sequence oligodeoxynucleotides.

The interaction of human DNA topoisomerase I (topo I) with specific sequence oligodeoxynucleotides (ODNs) of different length and structure has been investigated. All the ODNs used were shown to be effective enzyme inhibitors and to inhibit the topo I catalyzed relaxation of scDNA in a competitive manner. Among two DNA regions (A and B) required for topo I-mediated DNA cleavage, the former was found to display the higher affinity for the enzyme. The enzyme's affinity for ODNs corresponding to the scissile strand (five and nine nucleotide units in length) is about 2-4 orders of magnitude higher than that for non-specific ODNs of the same length. Topo I can efficiently recognize even extremely short specific ODNs containing only two or three bases (AGA and pAG, Ki = 15 and 60 microM, respectively): the sequence AAGA (Ki = 10 microM) is essential for tight DNA binding to topo I. The affinities of ODNs corresponding to the non-scissile strand are significantly lower. The ligand's affinity increases with its length. Additionally, about a ten-fold enhancement of specific sequence affinity occurs due to stable duplex formation during enzyme preincubation with ligands before addition of scDNA. We believe the possibility of using the short specific oligonucleotides and its derivatives as topoisomerase I-targeting drugs could not be excluded.

High affinity interaction of mouse DNA topoisomerase I with di- and trinucleotides corresponding to specific sequences of supercoiled DNA cleaved chain.

Recently mouse DNA topoisomerase I (topo) was shown to possess high affinity for a single-stranded AAGACTTAG nonanucleotide (K(i) = 2.0 microM) corresponding to the scissile strand of the minimal DNA duplex, which is necessary for cleavage of supercoiled DNA. In order to determine the most important part of the above sequence for the DNA recognition by topo, the interactions of the enzyme with a set of extremely short (2-5 nucleotides in length) oligonucleotides corresponding to different parts of the nonanucleotide have been investigated. The affinities of different oligonucleotides corresponding to the CTTAG part of the sequence (K(i) = 0.13-0.92 mM) were shown to be significantly lower than that for the AAGA tetranucleotide (K(i) = 9.0 microM). Topo effectively recognized even short oligonucleotides containing only two or three bases (AGA and pAG, K(i) = 20 and 50 microM). We suppose that oligonucleotides having a high afffinity to the enzyme can offer a unique opportunity for the rational design of topoisomerase-targeting drugs.

High affinity interaction of mammalian DNA topoisomerase I with short single- and double-stranded oligonucleotides.

The interaction of DNA topoisomerase I (topo I) with a set of single- and double-stranded oligonucleotides containing 5-27 mononucleotides was investigated. All single- and double-stranded oligonucleotides were found to inhibit competitively the supercoiled DNA relaxation reaction catalyzed by topo I. The enzyme affinity for specific sequence pentanucleotides of the scissile (GACTT, Ki = 2 microM) and non-cleaved chain (AAGTC, Ki = 110 microM) is about 2-4 orders of magnitude higher than that for non-specific oligonucleotides. This specific sequence affinity increases in several cases; lengthening of single-stranded oligonucleotides, formation of stable duplexes between complementary oligonucleotides and preincubation of the enzyme with ligands before addition of supercoiled DNA. We assume that oligonucleotides having a high affinity to the enzyme can offer a unique opportunity for rational design of topoisomerase-targeting drugs.