Triple-stranded Structure Research Articles

Pathogenic CANVAS-causing but not nonpathogenic RFC1 DNA/RNA repeat motifs form quadruplex or triplex structures

Biallelic expansions of various tandem repeat sequence motifs are possible in RFC1, encoding the DNA replication/repair protein RFC1, yet only certain repeat motifs cause cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS). CANVAS presents enigmatic puzzles: The pathogenic path for CANVAS is unknown, nor is it understood why some, but not all expanded motifs are pathogenic. The most common pathogenic repeat is (AAGGG)n•(CCCTT)n, while (AAAAG)n•(CTTTT)n is the most common non-pathogenic motif. While both intronic motifs can be expanded and transcribed, only r(AAGGG)n is retained in the mutant RFC1 transcript. We show that only the pathogenic form unusual nucleic acid structures. Specifically, DNA and RNA of the pathogenic d(AAGGG)4 and r(AAGGG)4 form G-quadruplexes in potassium solution. Non-pathogenic repeats did not form G-quadruplexes. Triple-stranded structures formed by the pathogenic motifs, but not by the non-pathogenic motifs. G- and C-richness of the pathogenic strands, (AAGGG)n•(CCCTT)n, favor formation of G•G•G•G-tetrads and protonated C+-G Hoogsteen base pairings, involved in quadruplex and triplex structures, respectively, each biophysically stabilized by increased hydrogen-bonds and pi-stacking interactions relative to A-T Hoogsteen pairs that could form by the non-pathogenic motif (AAAAG)n•(CTTTT)n. The quadruplex ligand, TMPyP4 binds the pathogenic quadruplexes. Formation of quadruplexes and triplexes by pathogenic repeats supports toxic-DNA and toxic-RNA modes of pathogenesis at the RFC1 gene and the RFC1 transcript. Our findings with short repeats provide insights into the disease specificity of pathogenic repeat motif sequences and reveal nucleic acid structural features that may be pathogenically involved and targeted therapeutically.

Topology and kinetics of R-loop formation

R-loops are structures containing an RNA-DNA duplex and an unpaired DNA strand. They can be formed upon “invasion” of an RNA strand into a DNA duplex, during which the RNA displaces the homologous DNA strand and binds the complementary strand. R-loops have many significant beneficial or deleterious biological effects, so it is important to understand the mechanisms for their generation and processing. We propose a model for co-transcriptional R-loop formation, in which their generation requires passage of the nascent RNA “tail” through the gap between the separated DNA strands. This passage becomes increasingly difficult with lengthening of the RNA tail. The length of the tail increases upon increasing distance between the transcription start site and the site of R-loop initiation. This causes reduced yields of R-loops with greater distance from the transcription start site. However, alternative pathways for R-loop formation are possible, involving either transient disruption of the transcription complex or the hypothetical formation of a triple-stranded structure, as a “collapsed R-loop.” These alternative pathways could account for the fact that in many systems R-loops are observed very far from the transcription start site. Our model is consistent with experimental data and makes general predictions about the kinetics of R-loop formation.

RNA:DNA triple helices: from peculiar structures to pervasive chromatin regulators.

The genomes of complex eukaryotes largely contain non-protein-coding DNA, which is pervasively transcribed into a plethora of non-coding RNAs (ncRNAs). The functional importance of many of these ncRNAs has been investigated in the last two decades, revealing their crucial and multifaceted roles in chromatin regulation. A common mode of action of ncRNAs is the recruitment of chromatin modifiers to specific regions in the genome. Whereas many ncRNA-protein interactions have been characterised in detail, binding of ncRNAs to their DNA target sites is much less understood. Recently developed RNA-centric methods have mapped the genome-wide distribution of ncRNAs, however, how ncRNAs achieve locus-specificity remains mainly unresolved. In terms of direct RNA-DNA interactions, two kinds of triple-stranded structures can be formed: R-loops consisting of an RNA:DNA hybrid and a looped out DNA strand, and RNA:DNA triple helices (triplexes), in which the RNA binds to the major groove of the DNA double helix by sequence-specific Hoogsteen base pairing. In this essay, we will review the current knowledge about RNA:DNA triplexes, summarising triplex formation rules, detection methods, and ncRNAs reported to engage in triplexes. While the functional characterisation of RNA:DNA triplexes is still anecdotal, recent advances in high-throughput and computational analyses indicate their widespread distribution in the genome. Thus, we are witnessing a paradigm shift in the appreciation of RNA:DNA triplexes, away from exotic structures towards a prominent mode of ncRNA-chromatin interactions.

Genomic profiling of native R loops with a DNA-RNA hybrid recognition sensor.

An R loop is a unique triple-stranded structure that participates in multiple key biological processes and is relevant to human diseases. Accurate and comprehensive R loop profiling is a prerequisite for R loops studies. However, current R loop mapping methods generate large discrepancies, therefore an independent method is in urgent need. Here, we establish an independent R loop CUT&Tag (Tn5-based cleavage under targets and tagmentation) method by combining CUT&Tag and GST-His6-2×HBD (glutathione S-transferase-hexahistidine-2× hybrid-binding domain), an artificial DNA-RNA hybrid sensor that specifically recognizes the DNA-RNA hybrids. We demonstrate that the R loop CUT&Tag is sensitive, reproducible, and convenient for native R loop mapping with high resolution, and find that the capture strategies, instead of the specificity of sensors, largely contribute to the disparities among different methods. Together, we provide an independent strategy for genomic profiling of native R loops and help resolve discrepancies among multiple R loop mapping methods.

Biophysical insights into the interaction of two enantiomers of Ru(II) complex [Ru(bpy)2(7-CH3-dppz)]2+ with the RNA poly(U-A⁎U) triplex.

To determine the factors affecting the stabilization of RNA triple-stranded structure by chiral Ru(II) polypyridyl complexes, a new pair of enantiomers, ∆-[Ru(bpy)2(7-CH3-dppz)]2+ (∆-1; bpy = 2,2'-bipyridine, 7-CH3-dppz = 7-methyl-dipyrido[3,2-a,2',3'-c]phenazine) and Λ-[Ru(bpy)2(7-CH3-dppz)]2+ (Λ-1), have been synthesized and characterized in this work. Binding properties of the two enantiomers with the RNA poly(U-A⁎U) triplex (where "-" denotes the Watson - Crick base pairing and "⁎" denotes the Hoogsteen base pairing) have been studied by spectroscopy and hydrodynamics methods. Under the conditions used in this study, changes in absorption spectra of the two enantiomers are not very different from each other when bound to the triplex, although the binding affinity of ∆-1 is higher than that of Λ-1. Fluorescence titrations and viscosity experiments give convincing evidence for a true intercalative binding of enantiomers with the triplex. However, melting experiments indicated that the two enantiomers selectively stabilized the triplex. The enantiomer ∆-1 stabilize the template duplex and third-strand of the triplex, while it's more effective for stabilization of the template duplex. In stark contrast to ∆-1, Λ-1 stabilizes the triplex without any effect on the third-strand stabilization, suggesting this one extremely prefers to stabilize the template duplex rather than third-strand. Besides, the triplex stabilization effect of ∆-1 is more marked in comparison with that of Λ-1. The obtained results suggest that substituent effects and chiralities of Ru(II) polypyridyl complexes play important roles in the triplex stabilization. Complexes Λ/Δ-[Ru(bpy)2(7-CH3-dppz)]2+ (Λ/Δ-1; bpy = 2,2'-bipyridine, 7-CH3-dppz = 7-methyl-dipyrido[3,2-a,2',3'-c]phenazine) were prepared as stabilizers for poly(U-A ∗ U) triplex. Results suggest the triplex stabilization depends the chiral structures of Λ/Δ-1, indicating that [Ru(bpy)2(7-CH3-dppz)]2+ is a non-specific intercalator for poly(U-A ∗ U) investigated in this work.

Formation of a DNA triple helical structure at BOLF1 gene of human herpesvirus 4 (HH4) genome

Eukaryotic genomes contain a large number of pyrimidine-purine rich regions and such regions can assume varied DNA conformations, including triple-stranded structures. These structures have fascinated scientists because of their considerable therapeutic applications. These structures have also profound implications in the field of nanotechnology as they can be used to develop DNA-based nanostructures and materials. Therefore, for any application, it is important to understand the formation of triplex structures, both in quantitative and qualitative terms. A combination of gel electrophoresis, UV-thermal denaturation and circular dichroism (CD) spectroscopy was used to investigate the formation of inter- as well as intramolecular triplex, in pyrimidine motif at BOLF1 gene of human herpesvirus 4 (HH4) genome. This gene codes for inner tegument protein, which plays crucial roles in viral replication. The said oligopurine•oligopyrimidine duplex was targeted via a designed triple helix forming oligopyrimidine nucleotide (TFO) in intermolecular as well as intramolecular fashion. Our studies revealed that intramolecular triplex formation takes place at acidic as well as at neutral pH; whereas low pH is required for its intermolecular version. This comparative study between inter- and intramolecular triplex allowed us to demonstrate that intramolecular structure is more stable to its intermolecular counterpart. Numerous models for mono-, bi- and trimolecular structures adopted by these DNA sequences have been suggested. This report adds to our existing knowledge about DNA triple helical structures.

Abstract 4838: R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells

Abstract G-quadruplexes (G4s) and R-loops are non-B DNA structures that can regulate basic processes such as transcription and replication. Unscheduled formation of R-loops is regarded as highly deleterious to cells, as R loops can induce replicative stress and DNA damage leading to genome instability. G4s are formed by four guanine residues held together by Hoogsteen hydrogen bonds and stabilized by monovalent cations. R loops are triple-stranded structures that contain an RNA-DNA hybrid duplex and a displaced single-stranded DNA. We have recently shown that Topoisomerase I (Top1) can affect genome-wide levels of R loops in human cancer cells consistently with the knowledge that DNA superhelical tension is a main driving force allowing non-B DNA structure formation. As G4 ligands were suggested to synergize with Top1 inhibitors, we asked the question of whether G4 ligands can affect R loops. We have thus investigated by immunofluorescence microscopy the effects of two different G4 binders, Pyridostatin (PDS) and FG (compound 1 in Amato et al J Med Chem 2016), on R-loops in human cancer cells. These G4 binders can increase both G4s and R loops while an inactive derivative of FG cannot induce them. Interestingly, the induction of G4s and R loops well correlate to each other in time and intensity with PDS being more effective than FG. After 24 hours, G4 binders induce a cell cycle arrest at the G2/M phase associated to double-stranded DNA cleavage as detected by γH2AX and 53BP1 foci formation and to activation of checkpoint response as shown by ATM phosphorylation. Interestingly, overexpression of RNaseH1 reduces both R loops and γH2AX foci induced by G4 binders showing that the ligands induce DNA cleavage via an R loop-mediated mechanism. Then, we silenced the BRCA2 gene by RNAi in U2OS cells and the results show that BRCA2 depletion increased γH2AX foci induced by PDS while overexpression of RNaseH1 rescue DNA cleavage induction. We also determined genomic R loop maps by DRIP-seq, and bioinformatic analyses of the specific location of G4-stabilized R loops provided information consistent with a model in which a G4 opposite to a DNA:RNA hybrid can stabilize R loops. Our study establishes for the first time that G4 binders stabilize either G4s and R loops in human cancer cells, and that they induce genome instability with a mechanism dependent on R loop formation. Partially supported by AIRC, Milan. Citation Format: Alessio De Magis, Stefano Giustino Manzo, Marco Russo, Olivier Sordet, Rita Morigi, Giovanni Capranico. R loop-driven genome instability by G-quadruplex binders in BRCA2-silenced human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4838.

A DNA Computing Model on Triple-Stranded for Minimum Spanning Tree Problem

Single-strand DNA can match with homologous double- stranded into a triple-stranded structure mediated by RecA protein.The paper provides a triple-stranded DNA computing model for minimum spanning tree problem. DNA fragments corresponding to edges are coded by double-stranded DNA, wrong hybridization does not take place and hairpin structure does not form. The single-strand DNA probe is bond with RecA protein, so the rate of wrong solution will reduce. And in this way, encoding complexity and the errors in computation will be decreased.

Systematic study of the synthesis and coordination of 2-(1,2,3-triazol-4-yl)-pyridine to Fe(II), Ni(II) and Zn(II); ion-induced folding into helicates, mesocates and larger architectures, and application to magnetism and self-selection.

With its facile synthesis, the pyridine-1,2,3-triazole chelate is an attractive building block for coordination-driven self-assembly. When two such chelates are bridged by a spacer and exposed to cations of octahedral geometrical preference, they generally self-assemble into dinuclear triple-stranded structures in the solid state and in solution in the presence of non-coordinating counter-ions. In solution, a wider range of architectures may nevertheless form, depending on the nature of the spacer. A systematic study of the spacer and substitution pattern is therefore presented, which allows assessing the various factors affecting self-assembly around the pyridine-1,2,3-triazole chelate, as well as the stereochemical control in these architectures. Applications to chirality, magnetism and system selection are discussed, and involve Fe(ii), Ni(ii), Zn(ii) and Cu(i) cations.

Effect of ancillary ligands on the interaction of ruthenium(II) complexes with the triplex RNA poly(U)·poly(A)*poly(U)

Two new Ru(II) complexes with 1,8-naphthalimide group, [Ru(phen)2(pnip)]2+ (Ru1; phen=1,10-phenanthroline, pnip=2-[N-(p-phenyl)-1,8-napthalimide]imidazo[4',5'-f][1,10]phenanthroline) and [Ru(bpy)2(pnip)]2+ (Ru2; bpy=2,2′-bipyridine), have been synthesized and characterized. The interactions of Ru1 and Ru2 with the triplex RNA poly(U)•poly(A)*poly(U) (where • denotes the Watson–Crick base pairing and * denotes the Hoogsteen base pairing) were studied by various biophysical. Electronic spectra established that the binding affinity for Ru1 was greater than that for Ru2. Fluorescence and viscosity studies gave convincing evidence for a true intercalative binding of both complexes with the RNA triplex. UV melting studies confirmed that the two complexes could stabilize the triplex, whereas the effects of the two complexes on the stability of the Hoogsteen base-paired strand ploy(U) and the Watson–Crick base-paired duplex poly(U)•poly(A) of the triplex were different. In the case of Ru1, the increase of the thermal stability of the Hoogsteen base-paired strand was stronger than that of the Watson–Crick base-paired duplex. However, an opposite effect was observed in the case of Ru2. Circular dichroic studies suggested that the RNA triplex undergoes a conformational transition in the presence of Ru1, whereas the helicity of the RNA triplex still remains A-type in the presence of Ru2. The main results obtained here further advance our knowledge on the interaction of RNA triple-stranded structures with metal complexes, particularly ruthenium(II) complexes.

Morphological Control of Helical Structures of an ABC-Type Triblock Terpolymer by Distribution Control of a Blending Homopolymer in a Block Copolymer Microdomain

The control of microphase-separated structures in a poly(styrene-block-butadiene-block-methyl methacrylate) (SBM) was investigated in three dimensions by transmission electron microtomography. Neat SBM self-assembled into a double-helical structure of polybutadiene (PB) domains around hexagonally packed core polystyrene (PS) cylinders in a poly(methyl methacrylate) matrix. When PS homopolymer with a lower molecular weight than that of the PS block in SBM was added to the SBM, the PB domains transformed from double-helical structures to spherical domains, while maintaining the helical trajectories. In contrast, adding a higher molecular weight PS to the SBM changed the helical structures from double- to triple-stranded structures and even to four-stranded structures. The helical structures of the PB domains were strongly affected by the distribution of the blended polystyrenes in the core cylindrical PS domains.

Crystal structure of (Gly‐Pro‐Hyp)9: Implications for the collagen molecular model

Collagens have long been believed to adopt a triple-stranded molecular structure with a 10/3 symmetry (ten triplet units in three turns) and an axial repeat of 29 Å. This belief even persisted after an alternative structure with a 7/2 symmetry (seven triplet units in two turns) with an axial repeat of 20 Å had been proposed. The uncertainty regarding the helical symmetry of collagens is attributed to inadequate X-ray fiber diffraction data. Therefore, for better understanding of the collagen helix, single-crystal analyses of peptides with simplified characteristic amino acid sequences and similar compositions to collagens have long been awaited. Here we report the crystal structure of (Gly-Pro-Hyp)(9) peptide at a resolution of 1.45 Å. The repeating unit of this peptide, Gly-Pro-Hyp, is the most typical sequence present in collagens, and it has been used as a basic repeating unit in fiber diffraction analyses of collagen. The (Gly-Pro-Hyp)(9) peptide adopts a triple-stranded structure with an average helical symmetry close to the ideal 7/2 helical model for collagen. This observation strongly suggests that the average molecular structure of collagen is not the accepted Rich and Crick 10/3 helical model but is a 7/2 helical conformation.

Anion-Dependent Formation of Helicates versus Mesocates of Triple-Stranded M2L3 (M = Fe2+, Cu2+) Complexes

A series of dinuclear triple-stranded complexes, [Fe(2)L(3)⊃X]X(6) [X = BF(4)(-) (1), ClO(4)(-) (2)], [Fe(2)L(3)⊃SO(4)](2)(SO(4))(5) (3), [Fe(2)L(3)⊃Br](BPh(4))(6) (4), Fe(2)L(3)(NO(3))Br(6) (5), and [Cu(2)L(3)⊃NO(3)](NO(3))(6) (6), which incorporate a central cavity to encapsulate different anions, have been synthesized via the self-assembly of iron(II) or copper(II) salts with the N,N'-bis[5-(2,2'-bipyridyl)methyl]imidazolium bromide (LBr) ligand. X-ray crystallographic studies (for 1-4 and 6) and elemental analyses confirmed the cagelike triple-stranded structure. The anionic guest is bound in the cage and shows remarkable influence on the outcome of the self-assembly process with regard to the configuration at the metal centers. The mesocates (with different configurations at the two metal centers) have formed in the presence of large tetrahedral anions, while helicates (with the same configuration at both metal centers) were obtained when using the relatively smaller spherical or trigonal-planar anions Br(-) or NO(3)(-).

The Molecular Interactions That Stabilize RNA Tertiary Structure: RNA Motifs, Patterns, and Networks

RNA molecules adopt specific three-dimensional structures critical to their function. Many essential metabolic processes, including protein synthesis and RNA splicing, are carried out by RNA molecules with elaborate tertiary structures (e.g. 3QIQ, right). Indeed, the ribosome and self-splicing introns are complex RNA machines. But even the coding regions in messenger RNAs and viral RNAs are flanked by highly structured untranslated regions, which provide regulatory information necessary for gene expression. RNA tertiary structure is defined as the three-dimensional arrangement of RNA building blocks, which include helical duplexes, triple-stranded structures, and other components that are held together through connections collectively termed RNA tertiary interactions. The structural diversity of these interactions is now a subject of intense investigation, involving the techniques of NMR, X-ray crystallography, chemical genetics, and phylogenetic analysis. At the same time, many investigators are using biophysical techniques to elucidate the driving forces for tertiary structure formation and the mechanisms for its stabilization. RNA tertiary folding is promoted by maximization of base stacking, much like the hydrophobic effect that drives protein folding. RNA folding also requires electrostatic stabilization, both through charge screening and site binding of metals, and it is enhanced by desolvation of the phosphate backbone. In this Account, we provide an overview of the features that specify and stabilize RNA tertiary structure. A major determinant for overall tertiary RNA architecture is local conformation in secondary-structure junctions, which are regions from which two or more duplexes project. At junctions and other structures, such as pseudoknots and kissing loops, adjacent helices stack on one another, and these coaxial stacks play a major role in dictating the overall architectural form of an RNA molecule. In addition to RNA junction topology, a second determinant for RNA tertiary structure is the formation of sequence-specific interactions. Networks of triple helices, tetraloop-receptor interactions, and other sequence-specific contacts establish the framework for the overall tertiary fold. The third determinant of tertiary structure is the formation of stabilizing stacking and backbone interactions, and many are not sequence specific. For example, ribose zippers allow 2'-hydroxyl groups on different RNA strands to form networks of interdigitated hydrogen bonds, serving to seal strands together and thereby stabilize adjacent substructures. These motifs often require monovalent and divalent cations, which can interact diffusely or through chelation to specific RNA functional groups. As we learn more about the components of RNA tertiary structure, we will be able to predict the structures of RNA molecules from their sequences, thereby obtaining key information about biological function. Understanding and predicting RNA structure is particularly important given the recent discovery that although most of our genome is transcribed into RNA molecules, few of them have a known function. The prevalence of RNA viruses and pathogens with RNA genomes makes RNA drug discovery an active area of research. Finally, knowledge of RNA structure will facilitate the engineering of supramolecular RNA structures, which can be used as nanomechanical components for new materials. But all of this promise depends on a better understanding of the RNA parts list, and how the pieces fit together.

Loop organization of eukaryotic chromosomes and triple-stranded DNA structures

To study possible involvement of polypurine and polypyrimidine DNA tracks potentially capable to form triple-stranded structures (H-form of DNA) in compactization of eukaryotic chromosomes a search in silico for "complementary" polypurine and polypyrimidine tracks within 12 eukaryotic gene nucleotide sequences was carried out. Polypurine and polypyrimidine tracks (10-11 b.p.) potentially capable of interacting with each other with the formation of triplex structures ("structurizing" regions) has been shown to be located in chromosomal locus of genes, predominantly in introns and flanking regions. In the case of in vivo realization of such DNA-DNA interactions the chromosomal gene domains can be folded into several small loops. An involvement of DNA triplexes in the chromosomal gene loci compactization may be associated with the gene functioning. The analogous analysis carried out for nucleotide sequences of long (LINE) and short (SINE) repeats dispersed over the genome as well as of satellite DNA has demonstrated a fundamental identity between mechanisms of chromosomal encoding and non-coding regions' compactization.

Friedreich's Ataxia Induced Pluripotent Stem Cells Model Intergenerational GAA⋅TTC Triplet Repeat Instability

The inherited neurodegenerative disease Friedreich's ataxia (FRDA) is caused by GAA⋅TTC triplet repeat hyperexpansions within the first intron of the FXN gene, encoding the mitochondrial protein frataxin. Long GAA⋅TTC repeats cause heterochromatin-mediated gene silencing and loss of frataxin in affected individuals. We report the derivation of induced pluripotent stem cells (iPSCs) from FRDA patient fibroblasts by transcription factor reprogramming. FXN gene repression is maintained in the iPSCs, as are the global gene expression signatures reflecting the human disease. GAA⋅TTC repeats uniquely in FXN in the iPSCs exhibit repeat instability similar to patient families, where they expand and/or contract with discrete changes in length between generations. The mismatch repair enzyme MSH2, implicated in repeat instability in other triplet repeat diseases, is highly expressed in pluripotent cells and occupies FXN intron 1, and shRNA silencing ofMSH2 impedes repeat expansion, providing a possible molecular explanation for repeat expansion in FRDA.

Performing DNA computation with RecA-mediated triple-stranded DNA structure

A new method of DNA computing using an unusual triple-stranded DNA structure mediated by RecA protein is introduced and a minor 3-variable instance of the satisfiability (SAT) problem is solved. Separations were performed by using biotinylated oligodeoxynucleotide probes coated with RecA protein. During the computation, sequence-specific recognition of double-stranded DNA by homologous oligodeoxynucleotides was fulfilled in the presence of ATPγS. Captured by the magnetic particles coated with streptavidin, the desirable double-stranded DNA strands were retained and extracted. It is suggested that the methods employed here may help decrease the errors in DNA computation and solve some larger size NP- complete problems.

A novel DNA computing model based on RecA-mediated triple-stranded DNA structure

Abstract The field of DNA computing emerged in 1994 after Adleman's paper was published. Henceforth, a few scholars solved some noted NP-complete problems in this way. And all these methods of DNA computing are based on conventional Watson-Click hydrogen bond of doublehelical DNA molecule. In this paper, we show that the triple-stranded DNA structure mediated by RecA protein can be used for solving computational problems. Sequence-specific recognition of double-stranded DNA by oligonucleotide-directed triple helix (triplex) formation is used to carry out the algorithm. We present procedure for the 3-vertex-colorability problems. In our proposed procedure, it is suggested that it is possible to solve more complicated problems with more variables by this model. *Supported by National Natural Science Foundation of China (Grant Nos. 60533010, 60373089, 60674106), Science Foundation of Shaanxi Bureau of Education (Grant No. 06JK202), Program for New Century Excellent Talents in University (No. NCET-05-0612) an...

RecA homologous DNA transferase: Functional activities and a search for homology by recombining DNA molecules

Bacterial RecA is a prototype of ATP-dependent homologous recombinases, found ubiquitously from bacteriophages to humans. The RecA filament formed on single-stranded DNA in the presence of ATP initiates a strand exchange reaction with homologous double-stranded DNA. Of the three stages of this reaction (search for homology, annealing of a triple-stranded structure accompanied by a switch of pairing, and displacement of the third strand), the first stage is the most enigmatic and least studied. As is generally accepted, this stage is directed by a special (extended) RecA filament structure and does not require any additional energy from ATP hydrolysis. The new approaches to the study of the strand exchange reaction with short oligonucleotides as DNA substrates and sensitive methods for a real-time monitoring of this reaction suggest that all three stages depend on ATP hydrolysis.

Specific Terminal Labeling of DNA Molecules

We report a simple method to directly label or modify a specific terminus of linear DNA molecules. The method is based upon our finding that a presumably triple-stranded structure by RecA-mediated formation at the terminus formed with deoxyoligonucleotides, whose sequence is complementary to the 5' terminus of one of the strands of a double-stranded DNA molecule, is quite stable and can serve as a template for DNA polymerase reaction, with the nucleotides being incorporated by an exchange reaction. This novel type of nucleotide incorporation has made it possible to label a specific terminus of target double-stranded DNA molecules by a direct means (without amplification) regardless of its molecular size, a procedure previously unavailable. As an application, we show that large DNA molecules can be fixed to a solid support in a specific orientation, thus being utilized for various analytical purposes of DNA molecules.