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All change at Holliday junction
Abstract
In the absence of added metal ions, the four-way junction is extended with an open central region (3, 4). However, upon addition of magnesium or other metal ions the junction folds by pairwise coaxial stacking of helices into the stacked X-structure (reviewed in refs. 5 and 6) (Fig. 1). This structure is a substrate for a variety of junction-selective enzymes, most of which alter the global structure on binding (reviewed in ref. 7). In free solution, the DNA junction folds to create an antiparallel structure, probably governed by the favorable juxtaposition of backbones and grooves when the small angle is approximately 60°. The lowering of symmetry on formation of the stacked X-structure creates two inequivalent kinds of strands; the two continuous strands turn about the pseudo-continuous axes that pass through the stacked helical pairs, while the exchanging strands pass between the stacked helices at the crossover. The general features of the stacked X-structure have been confirmed by a variety of experimental methods (3, 8–11 …
... Several bases at the loops are easily accessible to chemical probes and to single strand-speci®c enzymes, indicative of unpaired and unstacked bases (Sinden, 1994; Palecek, 1992). The bases at the junction are much less reactive, but they can be attacked by chemical probes at elevated temperatures or under conditions of low salt (McClellan & Lilley, 1987; Voloshin et al., 1989 ). Several studies using different physical chemical techniques have analyzed the structure of stable, immobile four-way DNA junctions (Cooper & Hagerman, 1987; Lilley & Clegg, 1993; Seeman & Kallenbach, 1994; Lilley, 1997). However, there has been little attempt to analyze the spatial geometry of cruciforms. ...
... Thus, cations stabilize the X-type cruciform structure rather than an extended one. These results are generally consistent with the effect of cations on geometry of immobile four-way junctions (reviewed by Cooper & Hagerman, 1987; Lilley & Clegg, 1993; Seeman & Kallenbach, 1994; Lilley, 1997): the junctions change from an extended conformation into the X-type geometry upon increasing concentrations of Mg or Na ions. To compare the geometric characteristics of the X-type cruciform at different ionic conditions, we measured the angles between the hairpins and between the main DNA strands for the images obtained under low salt conditions (TE buffer), high salt conditions (200 mM NaCl), and in the presence of Mg cations. ...
... At the same time, both sets of evidence favor our interpretation, namely that these two types of conformations of the cruciform, the compact X-type structure and an extended conformation, exist in solution. It is important that this interpretation as well as the stabilization of X-type conformation of the cruciform in high salt solutions is in accord with the data obtained for a rather similar system, immobile four-way junctions (Cooper & Hagerman, 1987; Eis & Millar, 1993; Lilley & Clegg, 1993; Seeman & Kallenbach, 1994; Lilley, 1997; Miick et al., 1997). Moreover, the AFM data indicate that the X-conformation has a strong bend in the main DNA strand, and this AFM observation is in remarkable coincidence with early gel retardation data (Gough & Lilley, 1985; Zheng & Sinden, 1988). ...
Understanding DNA function requires knowledge of the structure of local, sequence-dependent conformations that can be dramatically different from the B-form helix. One alternative DNA conformation is the cruciform, which has been shown to have a critical role in the initiation of DNA replication and the regulation of transcription in certain systems. In addition, cruciforms provide a model system for structural studies of Holliday junctions, intermediates in homologous DNA recombination. Cruciforms are not thermodynamically stable in linear DNA due to branch point migration, which makes their study using many biophysical techniques problematic. Atomic Force Microscopy (AFM) was applied to visualize cruciforms in negatively supercoiled plasmid DNA. Cruciforms are seen as clear-cut extrusions on the DNA filament with the lengths of the arms consistent with the size of the hairpins expected from a 106 bp inverted repeat. The cruciform exists in two different conformations, an extended one with the angle of ca. 180 degrees between the hairpin arms and a compact, X-type conformation, with acute angles between the hairpin arms and the main DNA strands. The ratio of molecules with the different conformations of cruciforms depends on ionic conditions. In the presence of high salt or Mg cations, a compact, X-type conformation is highly preferable. Remarkably, the X-conformation was highly mobile allowing the cruciform arms to adopt a parallel orientation. The structure observed is consistent with a model of the Holliday junction with a parallel orientation of the exchanging strands.
... A survey of sites cleaved by RusA, RuvC, and other resolvases, revealed that strand cleavage occurs in nearly every case between or next to a pyrimidine dinucleotide (Chan et al., 1997). Studies of the stacked X-structure adopted in the presence of metal ions showed that the sequence of base-pairs at the crossover determines the stacking preference of the duplex arms, and that the preferred stacking isomer maximises the number of purines in the exchanging strands forming acute angles at the crossover (Figure 5b; Azaro & Landy, 1997;Duckett et al., 1988Duckett et al., , 1995Lilley, 1997). It is therefore possible that some property of the pyrimidine dinucleotides found at resolution hotspots, or their purine complements, dictates the pattern of junction folding and resolution by RuvC. ...
... They are also identical in sequence outside of the central 4 bp. Bases in the second and third positions from the crossover are known to affect junction folding in the absence of protein (Lilley, 1997). ...
... Given the option of a purine or a pyrimidine in each case, base-speci®c contacts at these positions are unlikely. However, in the presence of metal ions, the bases at these positions could be important in establishing a precise fold at the crossover needed to establish speci®c contact with a T, or a TT (Lilley, 1997). Studies by H. Shinagawa and co-workers have shown that a highly conserved phenylalanine, Phe69, located at the catalytic centre ( Figure 7a) is critical for RuvC activity (Ariyoshi et al., 1994), and that while a tyrosine at this position allows resolution, a tryptophan does not (H. ...
The RuvC protein of Escherichia coli resolves Holliday intermediates in recombination and DNA repair by a dual strand incision mechanism targeted to specific DNA sequences located symmetrically at the crossover. Two classes of amino acid substitutions are described that provide new insights into the sequence-specificity of the resolution reaction. The first includes D7N and G14S, which modify or eliminate metal binding and prevent catalysis. The second, defined by G114D, G114N, and A116T, interfere with the ability of RuvC to cleave at preferred sequences, but allow resolution at non-consensus target sites. All five mutant proteins bind junction DNA and impose an open conformation. D7N and G14S fail to induce hypersensitivity to hydroxyl radicals, a property of RuvC previously thought to reflect junction opening. A different mechanism is proposed whereby ferrous ions are co-ordinated in the complex to induce a high local concentration of radicals. The open structure imposed by wild-type RuvC in Mg2+ is similar to that observed previously using a junction with a different stacking preference. G114D and A116T impose slightly altered structures. This subtle change may be sufficient to explain the failure of these proteins to cleave the sequences normally preferred. Gly114 and Ala116 residues link two alpha-helices lining the wall of the catalytic cleft in each subunit of RuvC. We suggest that substitutions at these positions realign these helices and interfere with the ability to establish base-specific contacts at resolution hotspots.
... A set of proteins was shown to participate in Holliday junction formation altering the topology of the DNA [21] enabling single-strand break appearance and approach of homologous DNA regions [22,23]. Synthetic Holliday junctions formed by half-complementary oligonucleotides have been shown to be cleaved both chemically [24,25] and by a number of enzymes [26,27]. It is in accordance with our proposal that background cleavage during CCM or EMC may be caused by the appearance of non-linear DNA structures in homologous duplex solution. ...
... The symmetry of all the x structures observed also confirmed that it was not overlapping. Our data on the different locations of the point of branch migration fit the theory that Holliday junctions cannot be viewed as stable structures but rather as an equilibrium mixture of conformational isomers [25,37,38] and that natural Holliday junctions with homologous sequence symmetry branch migrate to create a population of DNA structures that have the branch point at different sites. ...
Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA–DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA–protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.
... The cleavage step of this reaction is mediated by a four-way-junction-resolving enzyme typified by the E. coli RuvC protein [31,32]. An extruded cruciform has the essential features of a Holliday junction recombination intermediate that are required for recognition and processing by a junction-resolving enzyme [33][34][35][36]. Following DNA cleavage, joining by DNA ligase of nucleotides that were originally situated on opposite DNA strands results in linear products with covalently closed hairpin ends. ...
... No high-energy cofactor is required. In the pathway in (b), staggered DNA cleavages on opposing strands are introduced by endonucleolytic cleavage at the base of an extruded cruciform, as described for Holliday junction resolvases [31][32][33][34][35][36]. Dissociation of the complex brings the 5′ phosphate and the 3′ hydroxyl groups in proximity for ligase-mediated sealing of the nick to generate the closed hairpin products. ...
Linear DNA molecules with covalently closed hairpin ends (telomeres) exist in a wide variety of organisms. Telomere resolution, a DNA breakage and reunion reaction in which replicated telomeres are processed into hairpin ends, is now known to be a common theme in poxviruses, Borrelia burgdorferi and Escherichia coli phage N15. Candidate proteins that may perform this reaction have recently been identified in poxviruses. Moreover, the first purification and definitive identification of a telomere resolvase has been reported for phage N15. This protein is the prototype for a new class of DNA enzyme that performs a unique reaction. Advances in the study of telomere resolution in poxviruses, B. burgdorferi and E. coli phage N15 are discussed.
... Genetic recombination is the exchange of genetic material between different organisms which leads to production of offspring with combinations of traits that differ from those found in either parent and also goes through this junction intermediate. David Lilley showed that Mg 2+ ions determined the folding equilibria of synthetic junctions in solution and studied the process by FRET and by single molecule methods [40]. Junction formation clearly has specific charge neutralisation requirements, and in the closed form, which is Mg 2+ stabilised, the negative charges of the phosphate moieties are clearly brought close together. ...
84 years elapsed between the announcements of the periodic table and that of the DNA double helix in 1953, and the two have been combined in many ways since then. In this chapter an outline of the fundamentals of DNA structure leads into a range of examples showing how the natural magnesium and potassium ions found in nature can be substituted in a diversity of applications. The dynamic structures found in nature have been studied in the more controlled but artificial environment of the DNA crystal using examples from sodium to platinum and also in a range of DNA-binding metal complexes. While NMR is an essential technique for studying nucleic acid structure and conformation, most of our knowledge of metal ion binding has come from X-ray crystallography. These days the structures studied, and therefore also the diversity of metal binding, go beyond the double helix to triplexes, hairpin loops, junctions and quadruplexes, and the chapter describes briefly how these pieces fit into the DNA jigsaw. In a final section, the roles of metal cations in the crystallisation of new DNA structures are discussed, along with an introduction to the versatility of the periodic table of absorption edges for nucleic acid structure determination.
... Staples run along a single cylinder/helix for several bp and then cross over to a neighboring cylinder/helix making a ''u-turn'' in the process. The resulting u-turn cross-over geometry is termed a ''Holliday Juntion,'' and occurs in several physiological DNA structures [33]. Several of these Holliday junction cross-overs are spaced out along the length of the object. ...
This research introduces DNA origami as a viable approach to design and fabricate nanoscale mechanisms and machines. DNA origami is a recently developed nanotechnology that has enabled the construction of objects with unprecedented nanoscale geometric complexity via self-assembly. These objects are made up of thousands of DNA base-pairs packed into 3D structures with typical dimensions of 10–100nm. The majority of DNA origami research to date focuses on assembly of static 2D or 3D structures. In this work, we aim to extend the scope of DNA origami to include design of objects with kinematically constrained moving parts. Borrowing concepts from macro-scale kinematic mechanisms, we propose the concept of DNA Origami Mechanisms and Machines (DOMM) comprised of multiple links connected by joints. The links are designed by bundling double stranded DNA (dsDNA) helices to achieve the desired geometry and stiffness. The joints are designed by combining links with strategic placement flexible single stranded DNA (ssDNA) to enable motion in specific degrees of freedom. We detail design approaches for links and common joints including revolute, prismatic, and spherical, and discuss their integration into higher order mechanisms. As a proof of concept, we built a nanoscale hinge (revolute joint) and integrated four of these hinges into a prototype DOMM, namely a Bennett 4-bar linkage, which can be completely folded into a closed bundle geometry and unfolded into an open square geometry with a specified kinematic motion path. A kinematic analysis shows that the DNA Bennett linkage closely follows the 3D motion path of the rigid body counterpart. Our results demonstrate that DNA origami has high potential for the design and assembly of nanoscale machines. The ultimate goal of this work is to develop a library of nanoscale DNA-based links and joints that can be widely used in the design and assembly of higher order mechanisms and machines. We anticipate that, in the future, these components can be used to build nanorobots for useful applications including drug delivery, nanomanufacturing, and biosensing.
... This separation of Holliday junctions is performed by resolvase proteins such as GEN1 (Ip et al., 2008) that specifically recognise and bind to four-way junctions, again sequence-independently. Aside from proteins involved in recombination, HMG1 box proteins, SWI/SNF complex and unrelated prokaryotic proteins also recognise and bind to four-way junctions (Lilley, 1997). The ability to interact with bent DNA and capacity to increase DNA bending following binding (Zlatanova and van Holde, 1998) are common properties of such proteins. ...
... In recent years, it has been found that many variations from the Watson-Crick duplex structure [1] play key roles in many cellular processes. Examples are hairpins, [2] cruciforms, [3] parallel-stranded duplexes, [4] triplexes, [5] G-quadruplexes, [6] and the i-motif. [7] These structures can be formed by nucleotide sequences distributed throughout the whole human genome, their location is not random and often associated with human diseases. ...
Triplex als Alternative: Metadynamikrechnungen deuten darauf hin, dass das Thrombin bindende Aptamer (TBA) neben dem üblichen G‐Quadruplex auch eine stabile G‐Triplex‐Struktur einnehmen kann (siehe Schema; rote Kugel: K+‐Ion). Ein 11‐mer‐Oligonucleotid bildet ebenfalls einen stabilen G‐Triplex, dessen Struktur und thermodynamische Eigenschaften charakterisiert wurden.
... Staples run along a single cylinder/helix for several bp and then cross over to a neighboring cylinder/helix making a ''u-turn'' in the process. The resulting u-turn cross-over geometry is termed a ''Holliday Juntion,'' and occurs in several physiological DNA structures [33]. Several of these Holliday junction cross-overs are spaced out along the length of the object. ...
The goal of this paper is to introduce scaffolded DNA origami as a viable approach to the design of nanoscale mechanisms and machines. Resembling concepts of links and joints in macro scale mechanisms and machines, we propose the concept of DNA Origami Mechanisms and Machines (DOMM) that are comprised of multiple links connected by joints. Realization of nanoscale machines would pave the way for novel devices and processes with potential to revolutionize medicine, manufacturing, and environmental sensing. The realization of nanoscale machines and robots will enable scientists to manipulate and assemble nano objects in a more precise, efficient and convenient way at the molecular scale. For example, DNA nanomachinery could potentially be used for nano manufacturing, molecular transport in bioreactors, targeting cancer cells for drug delivery, or even repairing damaged tissue. As a proof of concept, we build a nanoscale spatial Bennett 4-bar mechanism that can be completely folded and unfolded with a specified kinematic motion path. The links comprise a 16 double stranded DNA (dsDNA) helices bundled in a 4 by 4 square cross-section yielding a high mechanical stiffness. The joints (in this case hinges) are designed using single strand DNA (ssDNA) connections between the links. This DOMM was designed within caDNAno, a recently developed computer-aided DNA origami design software, and then fabricated via a molecular self-assembly process. The resulting structure was imaged by transmission electron microscopy to identify structural conformations. Our results show that the designed DNA origami Bennett mechanism closely follows the kinematics of their rigid body counterparts. This research has the potential of opening a new era of design, analysis and manufacture of nanomechanisms, nanomachines and nanorobots.
... of the nucleobases (syn and anti) (Figure 1.14), and contains only one deep helical groove. at low pH), quadruplexes (by folding of a guanosine-rich single chain), and Holliday junctions (of four DNA strands), [126,130,131] important for interaction with biological components, such as proteins. By comparison to DNA, RNA possesses higher structural and dynamic flexibility and has a higher the propensity to fold and form 3D higherordered structures that alternate helices and single-stranded regions or loops. ...
The fascinating way nature relies on biomolecules, mostly proteins and sometimes RNA, to carry out sophisticated chemical processes led to more and more efforts to use the concepts of biology for preparing efficient chiral catalysts. The hybrid catalyst approach that combines the steric information derived from a protein scaffold with the catalytic activity of transition metal complexes offers a resourceful means of developing semisynthetic metalloenzymes for enantioselective applications. Since the discovery of nucleic acids with enzyme-like functions, the catalytic potential of nucleic acids is being revealed by in vitro selection and evolution of novel ribozymes and DNAzymes. Nucleic acids, especially RNA, appear to be versatile catalysts capable of accelerating a broad range of reactions and exquisitely discriminating between chiral targets. However, while proteins dominated the construction of hybrid catalysts, the application of DNA and RNA in asymmetric catalysis has hardly been explored. This work aimed at exploring the chirality of nucleic acids and generating hybrid catalysts based on DNA and RNA. Towards the development of metallo-(deoxy)ribozymes assisted by combinatorial strategies (e.g., SELEX), a straightforward synthetic way of embedding transition metal complexes in nucleic acids folds was established. DNA sequences carrying mono- and bidentate phosphine ligands as well as P,N-ligands were successfully prepared starting from amino-modified oligonucleotide precursors. The optimized convertible nucleoside approach allowed the parallel, high-yielding synthesis of various alkylamino-DNA conjugates differing in length and structure of the spacer. Coupling of amino-oligonucleotides with PYRPHOS, BINAP and PHOX ligands equipped with a carboxyl group led to the incorporation of phosphine moieties at predetermined internal sites. Moreover, the stability of the DNA-tethered BINAP and PHOX was reasonably high, which makes them attractive candidates for the development of transition metal-containing oligonucleotides. To this end, systematic studies on the behavior of phosphine- and PHOX-metal complexes in aqueous medium - a prerequisite of nucleic acid catalysts - were carried out. Two model organometallic transformations were selected that were compatible with the structure and chemistry of nucleic acids. The rhodium(I)-catalyzed 1,4-addition of phenyl boronic acid to 2-cyclohexen-1-one and iridium(I)-catalyzed allylic amination of the branched phenyl allyl acetate, respectively, proceeded efficiently in the presence of phosphorus-based ligands, in aqueous medium, at room temperature and low catalyst concentration. For the first model reaction, the best conversion (80%) was achieved with the isolated [Rh(nbd)BINAP]BF4 complex, in 6:1 dioxane/water, and TEA additive. On the basis of these data, a suitable system for assessing the catalytic potential of the DNA-BINAP ligand was implemented. In the second chosen reaction the in situ formed Ir(I)-PHOX complexes (0.05-0.1 mM) gave rise to racemic, branched allylic amination products in good yields (33-75%), in 3:7 dioxane/water. Kinetic resolution of the racemic substrate was then attempted by employing catalysts generated from the [Ir(cod)Cl]2 precursor and single- and double-stranded DNA-PHOX conjugates. Good conversions were obtained in the presence of G-poor DNA/DNA and RNA/DNA hybrids bearing the PHOX moiety, indicating a potential role of the G-N7 site in the first coordination sphere. With all tested DNA-PHOX conjugates, the levels of enantioselectivity remained modest. The results described in this work provide useful information for understanding the influence of nucleic acid sequence and covalent tethering on the reaction outcome. These are the first reported applications of DNA-based ligands in organometallic catalysis and they build the fundamentals for further development of selective nucleic acid catalysts, by means of rational design and in vitro selection approaches. Die faszinierende Art und Weise, in der die Natur auf Biomoleküle - meist Proteine und teilweise RNA - zurückgreift um anspruchsvolle chemische Prozesse auszuführen, hat zu immer mehr Bemühungen geführt, die Konzepte der Biologie für die Herstellung effizienter chiraler Katalysatoren zu nutzen. Die Verbindung dreidimensionaler Proteinstrukturen mit der katalytischen Aktivität von Übergangsmetallkomplexen ist eine interessante Herangehensweise in der Synthese sogenannter Hybrid-Katalysatoren für enantioselektiven Anwendungen. Seit der Entdeckung von Nukleinsäuren mit enzym-ähnlichen Funktionen wurde deren katalytisches Potential durch in vitro Selektion und Evolution neuer Ribozyme und DNAzyme deutlich gezeigt. Nukleinsäuren, insbesondere RNA, sind demnach vielseitige Katalysatoren, die in der Lage sind eine breite Palette an Reaktionen zu beschleunigen und außerordentlich gut zwischen chiralen Zielmolekülen zu unterscheiden. Während Proteine die Entwicklung von Hybrid- Katalysatoren jedoch weitgehend beherrschen, wurde die Anwendung von DNA oder RNA in der asymmetrischen Katalyse bisher kaum untersucht. Das Ziel dieser Arbeit war die Synthese von Hybrid-Katalysatoren auf Basis eines DNA und RNA Gerüstes. Für die kombinatorisch-gestützte Entwicklung von Metallo-Ribozymen und -Deoxyribozymen (z. B. mittels SELEX) wurde ein direkter Syntheseweg zum Einbau von Übergangsmetall-Komplexen in Nukleinsäurestrukturen etabliert. DNA Sequenzen welche ein- und zweizähnige Phosphin-Liganden sowie P,N-Liganden tragen, wurden ausgehend von amino-modifizierten Oligonukleotid Vorstufen erfolgreich synthetisiert. Der optimierte Ansatz mittels sogenannter convertable nucleosides erlaubt die parallele Synthese verschiedener alkylamino-DNA Konjugate, welche sich in Länge und Struktur der Spacer unterscheiden. Die Kopplung der Amino-Oligonukleotide mit PYRPHOS-, BINAP- und PHOX-Liganden, welche mit einer Carboxylgruppe ausgestattet sind, führt zum Einbau der Phosphin Bausteine an einer zuvor festgelegten Stelle im Nukleotidstrang. Ferner macht die hohe Stabilität der DNA-gebundenen BINAP und PHOX Liganden diese zu attraktiven Kandidaten für die Entwicklung von Oligonukleotiden, die Übergangsmetall-Komplexe enthalten. Zu diesem Zweck wurden systematische Studien zum Verhalten von Phosphin- und PHOX-Metallkomplexen im wässrigen Medium durchgeführt - eine Voraussetzung für Katalysatoren auf Nukleinsäurebasis. Zwei metallorganische Transformationen, die mit der Struktur und den chemischen Eigenschaften von Nukleinsäuren kompatibel sind, wurden als Modellreaktionen ausgewählt. Die Rhodium(I)-katalysierte 1,4-Addition von Phenylborsäure zu 2-Cyclohexen-1-on und die Iridium(I)-katalysierte allylische Aminierung des verzweigten Phenylallylacetats verliefen erfolgreich in Anwesenheit von phosphorbasierten Liganden, in wässrigem Medium, Raumtemperatur und niedriger Katalysatorkonzentration. Für die erste Modellreaktion wurde die beste Umsetzung mit dem isolierten [Rh(nbd)BINAP]BF4 Komplex in 6:1 Dioxan/Wasser und unter TEA Zugabe erzielt (80 %). Auf Grundlage dieser Daten wurde ein geeignetes System erstellt, um das katalytische Potential von DNA-BINAP Liganden zu beurteilen. Bei der zweiten Modellreaktion führte der in situ gebildete Ir(I)-PHOX Komplex (0.05-0.1 mM) in 3:7 Dioxan/Wasser zu guten Ausbeuten (33-75 %) der racemischen, verzweigten Aminierungsprodukte. Bei der kinetischen Auflösung racemischer Substrate wurden Katalysatoren verwendet, die aus dem [Ir(cod)Cl]2-Vorstufe und einfach- und doppelsträngigen DNA-PHOX Konjugaten hergestellt wurden. Gute Umsätze wurden in Anwesenheit von G-armen DNA/DNA und RNA/DNA Hybriden, die eine PHOX Gruppe tragen, erzielt, was auf eine mögliche Funktion der G-N7 Position in der ersten Koordinationsspähre hindeutet. Die Enatioselektivität blieb jedoch bei allen getesteten DNA-PHOX Konjugaten gering. Die Ergebnisse dieser Arbeit bieten hilfreiche Informationen für das Verständnis darüber, welchen Einfluss die Nukleinsäuresequenz und die kovalente Verknüpfung auf den Ausgang der Reaktion haben. Dies ist der erste Bericht über die Anwendungen von DNA-basierten Liganden in der metallorganischen Katalyse und setzt den Grundstein für die weitere Entwicklung selektiver Nukleinsäurekatalysatoren mittels der Methodik des rationalen Designs und der in vitro Selektion.
... To date, structures of four members of the integrase family have been solved by Xray crystallography: the Int proteins from phages HP1 and l, XerD from Escherichia coli and Cre from phage P1 (Guo et al., 1997;Hickman et al., 1997;Kwon et al., 1997;Subramanya et al., 1997). Considering their divergent sequences, these proteins show remarkable conservation of overall structure and nearly superimposable catalytic sites (for reviews, see Grindley, 1993;Lilley, 1997;Yang & Mizuuchi, 1997). The active-site geometry is also very similar to that of type IB topoisomerases (for reviews, see Nash, 1998;Sherratt & Wigley, 1998) which utilize a similar chemical mechanism of cleavage and ligation steps as the tyrosine recombinases. ...
The Flp site-specific recombinase functions in the copy number amplification of the yeast 2 microm plasmid. The recombination reaction is catalyzed by four monomers of Flp bound to two separate, but identical, recombination sites (FRT sites) and occurs in two sequential pairs of strand exchanges. The relative orientation of the two recombination sites during synapsis was examined. Topoisomerase relaxation and nick ligation were used to detect topological nodes introduced by the synapse prior to the chemical steps of recombination. A single negative supercoil was found to be trapped by Flp in substrates with inverted FRT sites whereas no trapped supercoils were observed with direct repeats. The topology of products resulting from Flp-mediated recombination adjacent to a well characterised synapse, that of Tn3 resolvase/res, was analyzed. The deletion and inversion reactions yielded the four noded catenane and the three noded knot, respectively, as the simplest and the most abundant products. The linking number change introduced by the Flp-mediated inversion reaction was determined to be +/-2. The most parsimonious explanation of these results is that Flp aligns its recombination sites with antiparallel geometry. The majority of synapses appear to occur without entrapment of additional random plectonemic DNA supercoils between the sites and no additional crossings are introduced as a result of the chemical steps of recombination.
... Structural studies of the stacked configuration are relatively rare though X-ray, NMR [14] and simulation [15] techniques have revealed likely binding site locations [16]. Most of the interest in the structure and properties of branched DNA intermediates arises from their importance in genetic recombination processes [6], recognition and processing by proteins [17], viral integration [18] and DNA repair [19]. ...
A Holliday junction (HJ) consists of four DNA double helices, with a branch point discontinuity at the intersection of the component strands. At low ionic strength, the HJ adopts an open conformation, with four widely spaced arms, primarily due to strong electrostatic repulsion between the phosphate groups on the backbones. At high ionic strength, screening of this repulsion induces a switch to a more compact (closed) junction conformation. Fluorescent labelling with dyes placed on the HJ arms allows this conformational switch to be detected optically using fluorescence resonance energy transfer (FRET), producing a sensitive fluorescent output of the switch state. This paper presents a systematic and quantitative survey of the switch characteristics of such a labelled HJ. A short HJ (arm length 8 bp) is shown to be prone to dissociation at low switching ion concentration, whereas an HJ of arm length 12 bp is shown to be stable over all switching ion concentrations studied. The switching characteristics of this HJ have been systematically and quantitatively studied for a variety of switching ions, by measuring the required ion concentration, the sharpness of the switching transition and the fluorescent output intensity of the open and closed states. This stable HJ is shown to have favourable switch characteristics for a number of inorganic switching ions, making it a promising candidate for use in nanoscale biomolecular switch devices.
... of helices into the stacked X structure that is unable to Forks progressively invade adjacent regions as shown efficiently branch migrate (Duckett et al., 1988(Duckett et al., , 1990; by the appearance of the Y arc on restriction fragments Lilley, 1997). We therefore tested whether the branch A-D. ...
Cells overcome intra-S DNA damage and replication impediments by coupling chromosome replication to sister chromatid-mediated recombination and replication-bypass processes. Further, molecular junctions between replicated molecules have been suggested to assist sister chromatid cohesion until anaphase. Using two-dimensional gel electrophoresis, we have identified, in yeast cells, replication-dependent X-shaped molecules that appear during origin activation, branch migrate, and distribute along the replicon through a mechanism influenced by the rate of fork progression. Their formation is independent of Rad51- and Rad52-mediated homologous recombination events and is not affected by DNA damage or replication blocks. Further, in hydroxyurea-treated rad53 mutants, altered in the replication checkpoint, the branched molecules progressively degenerate and likely contribute to generate pathological structures. We suggest that cells couple sister chromatid tethering with replication initiation by generating specialized joint molecules resembling hemicatenanes: this process might prime cohesion and assist sister chromatid-mediated recombination and replication events.
The grooves of DNA provide recognition sites for many nucleic acid binding proteins and anticancer drugs such as the covalently binding cisplatin. Here we report a crystal structure showing, for the first time, groove selectivity by an intercalating ruthenium complex. The complex Λ‐[Ru(phen)2phi]2+, where phi = 9,10‐phenanthrenediimine, is bound to the DNA decamer duplex d(CCGGTACCGG)2. The structure shows that the metal complex is symmetrically bound in the major groove at the central TA/TA step, and asymmetrically bound in the minor groove at the adjacent GG/CC steps. A third type of binding links the strands, in which each terminal cytosine base stacks with one phen ligand. The overall binding stoichiometry is four Ru complexes per duplex. Complementary biophysical measurements confirm the binding preference for the Λ‐enantiomer and show a high affinity for TA/TA steps and, more generally, TA‐rich sequences. A striking enantiospecific elevation of melting temperatures is found for oligonucleotides which include the TATA box sequence.
The grooves of DNA provide recognition sites for many nucleic acid binding proteins and anticancer drugs such as the covalently binding cisplatin. Here we report a crystal structure showing, for the first time, groove selectivity by an intercalating ruthenium complex. The complex Λ‐[Ru(phen)2phi]²⁺, where phi=9,10‐phenanthrenediimine, is bound to the DNA decamer duplex d(CCGGTACCGG)2. The structure shows that the metal complex is symmetrically bound in the major groove at the central TA/TA step, and asymmetrically bound in the minor groove at the adjacent GG/CC steps. A third type of binding links the strands, in which each terminal cytosine base stacks with one phen ligand. The overall binding stoichiometry is four Ru complexes per duplex. Complementary biophysical measurements confirm the binding preference for the Λ‐enantiomer and show a high affinity for TA/TA steps and, more generally, TA‐rich sequences. A striking enantiospecific elevation of melting temperatures is found for oligonucleotides which include the TATA box sequence.
DNA replication is arguably the most important biological process associated with cancer progression. This is evident as the inhibition of DNA synthesis remains one of the key therapeutic strategies used to treat this disease. This chapter describes the biological process of DNA replication focusing primarily on the roles of DNA polymerases in cancer progression and chemotherapy. The function of human DNA polymerases in replication, repair, recombination, and translesion DNA synthesis (TLS) are discussed with a special emphasis on their biochemical, structural, and mechanistic features. The current arsenal of therapeutic agents used to inhibit DNA polymerase activity is described, paying particular attention to purine and pyrimidine nucleoside analogs. Preclinical and clinical applications of these nucleoside analogs are described with respect to mono- and combination therapy using DNA-damaging agents such as chlorambucil and cisplatin.
This chapter discusses the chemotherapy intervention by inhibiting DNA polymerases. A fundamental feature of all cancers is their hyperproliferative nature that is defined by uncontrollable and, in some cases, pro-mutagenic DNA replication. DNA replication is the process by which genetic information is duplicated to produce two identical copies of an organism's genome. All DNA polymerases share a common mechanism for DNA chain synthesis that involves the covalent linking of one nucleotide at a time to the end of a preexisting DNA chain serving as a primer. Since the sequence of the template varies, DNA polymerases are faced with the difficult task of remaining flexible enough to recognize four distinct pairing combinations while being stringent enough to maintain faithful duplication of the template so that A is always incorporated opposite T and never opposite C, G, or A.
The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence
of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases.
In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase,
highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with
an apparent dissociation constant (Kd) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic
Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg2+ at 5–10 mM was optimal. Mn2+ could substitute for Mg2+, but it was much less efficient than Mg2+ as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at ∼200 mM. In addition to the high specific
activity, Hjc was found to be extremely heat stable. In contrast to the case of Sulfolobus, the Holliday junction resolving activity detected in P.furiosus cell extract thus far is only derived from Hjc.
Affinity chromatography using oligo DNA from a cis-active region (-453/-389) of the human DSC2 promoter was used to isolate a protein from human CaCo-2 epithelial cells expressing DSC2. The protein was SDS-PAGE purified, trypsinized and analyzed by MALDI-TOF mass spectrometry. Peptide mass fingerprinting identified the protein as nucleolin, a ubiquitously expressed protein involved in gene regulation.
Triplex with a twist: Through metadynamics calculations, the thrombin binding aptamer (TBA) has been shown to adopt a stable G-triplex structural motif, in addition to the usual G-quadruplex. An 11-mer oligonucleotide was also shown to form a stable G-triplex, whose structural and thermodynamic properties have been characterized.
Previously, using concentrated solutions of PCR products of five different genes, we described the appearance in these solutions of DNA structures with molecular weights approximately twice greater than that of double-strand (ds) fragments and with even higher molecular weight. Since this phenomenon was shown to be not dependent on the size or sequence of the DNA fragments, we suggested that it is due to interaction of DNA duplexes. The double-sized dsDNA complex containing four polynucleotide strands of two DNA fragments was named a "tetramer". Our present work is devoted to elucidation of peculiarities of tetramer formation and its structure in solutions of a purified PCR product of p53 cDNA. We found that the intensity of tetramer formation depends on the concentration of the PCR product in solution. Three subsequent purifications of the PCR product were performed using DNA-binding matrix, but the tetramers appeared again after every procedure. After purification of PCR product preliminarily treated with S1-nuclease, tetramers appeared again, indicating that these structures are formed from dsDNA fragments. Purification of the tetramers on DNA-binding matrix led to the appearance of the initial dsDNA fragments as the main DNA structure. When electroelution and column filtration by centrifugation were used, the purification procedure was speeded up, and a solution with a higher amount of the tetramer was obtained. Electron microscopy revealed the presence of four-stranded symmetrical structures with crossing chains known as Holliday junctions. Thus, for the first time the ability of homologous dsDNA fragments to interact with the formation of Holliday junctions without participation of cell proteins has been demonstrated.
Proteins that can be shown to strongly bind in vitro to the four-way (Holliday) junction DNA include not only the obvious candidates such as enzymes involved in recombination, but also a remarkably diverse group of seemingly unrelated proteins. These include the HMG1 box proteins, members of the HMGI-Y family, winged helix proteins (including linker histones), the SWI/SNF complex, and some totally unrelated prokaryotic proteins. What these proteins seem to share is a propensity to bind to bent DNA, to bend DNA upon binding, and/or to preferentially interact with DNA crossings. Thus, they appear to be, in the main, architectural proteins, although some (like the SWI/SNF complex) have very specific functional roles as well. Perhaps because they bind to or promote the formation of particular DNA structures, the four-way junction binding proteins are frequently interchangeable in cellular function. Furthermore, since a given kind of structure can be recognized by many different protein motifs, it is not surprising that apparently unrelated proteins can fall into such a single functional class.
CCE1 is a Holliday (four-way DNA) junction-specific endonuclease which resolves mitochondrial DNA recombination intermediates in Saccharomycescerevisiae. The junction-resolving enzymes are a diverse class, widely distributed in nature from viruses to higher eukaryotes. In common with most other junction-resolving enzymes, the cleavage activity of CCE1 is nucleotide sequence-dependent. We have undertaken a systematic study of the sequence specificity of CCE1, using a single-turnover kinetic assay and a panel of synthetic four-way DNA junction substrates. A tetranucleotide consensus cleavage sequence 5'-ACT downward arrowA has been identified, with specificity residing mainly at the central CT dinucleotide. Equilibrium constants for CCE1 binding to four-way junctions are unaffected by sequence variations, suggesting that substrate discrimination occurs predominantly in the transition state complex. CCE1 cuts most efficiently at the junction center, but can also cleave the DNA backbone at positions one nucleotide 3' or 5' of the point of strand exchange, suggesting a significant degree of conformational flexibility in the CCE1:junction complex. Introduction of base analogues at single sites in four-way junctions has allowed investigation of the sequence specificity of CCE1 in finer detail. In particular, the N7 moiety of the guanine base-pairing with the cytosine of the consensus sequence appears to be crucial for catalysis. The functional significance of sequence specificity in junction-resolving enzymes is discussed.
Histone H1, HMG-1 and HMG-I(Y) are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both HMG-1 and histone H1 on 4H DNA and compared these contacts with those previously described for HMG-I(Y) on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the HMG-I(Y) and HMG-1 proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of histone H1 bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized HMG-I(Y) protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of HMG-I(Y), histone H1 and HMG-1 in biological processes such as genetic recombination and retroviral integration.
Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae. Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange. In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques. Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer. Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium. The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold. This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments [Overmars and Altona (1997) J. Mol. Biol. 273, 519-524; Panyutin et al. (1995) EMBO J. 14, 1819-1826]. The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.
The rearrangement and repair of DNA by homologous recombination often involves the creation of Holliday junctions, which must be cleaved by junction-specific endonucleases to yield recombinant duplex DNA products. Holliday junction resolving enzymes are a ubiquitous class of proteins with diverse structural and mechanistic characteristics. We have characterised an endonuclease (Hje) from the thermophilic crenarchaeote Sulfolobus solfataricus that exhibits a high degree of specificity for Holliday junctions via an apparently novel mechanism. Hje resolves four-way DNA junctions by the introduction of paired nicks in a reaction that is independent of the local nucleotide sequence, but is restricted solely to strands that are continuous in the stacked-X form of the junction. Three-way DNA junctions are cleaved only when the presence of a bulge in one strand allows the junction to stack in an analogous manner to four-way junctions. These properties differentiate Hje from all other known junction resolving enzymes.
We present a simple theory of the dynamics of force generation by RecA during homologous strand exchange and a continuous,
deterministic mathematical model of the proposed process. Calculations show that force generation is possible in this model
for certain reasonable values of the parameters. We predict the shape of the force-velocity curve for the Holliday junction,
which exhibits a distinctive kink at large retarding force, and suggest experiments which should distinguish between the proposed
model and other models in the literature.
Holliday junction resolving enzymes are ubiquitous proteins that function in the pathway of homologous recombination, catalyzing
the rearrangement and repair of DNA. They are metal ion-dependent endonucleases with strong structural specificity for branched
DNA species. Whereas the eukaryotic nuclear enzyme remains unknown, an archaeal Holliday junction resolving enzyme, Hjc, has
recently been identified. We demonstrate that Hjc manipulates the global structure of the Holliday junction into a 2-fold
symmetric X shape, with local disruption of base pairing around the point of cleavage that occurs in a region of duplex DNA
3′ to the point of strand exchange. Primary and secondary structural analysis reveals the presence of a conserved catalytic
metal ion binding domain in Hjc that has been identified previously in several restriction enzymes. The roles of catalytic
residues conserved within this domain have been confirmed by site-directed mutagenesis. This is the first example of this
domain in an archaeal enzyme of known function as well as the first in a Holliday junction resolving enzyme.
The Hjc protein of Pyrococcus furiosus is an endonuclease that resolves Holliday junctions, the intermediates in homologous recombination. The amino acid sequence of Hjc is conserved in Archaea, however, it is not similar to any of the well-characterized Holliday junction resolvases. In order to investigate the similarity and diversity of the enzymatic properties of Hjc as a Holliday junction resolvase, highly purified Hjc produced in recombinant Escherichia coli was used for detailed biochemical characterizations. Hjc has specific binding activity to the Holliday-structured DNA, with an apparent dissociation constant (K:(d)) of 60 nM. The dimeric form of Hjc binds to the substrate DNA. The optimal reaction conditions were determined using a synthetic Holliday junction as substrate. Hjc required a divalent cation for cleavage activity and Mg(2+) at 5-10 mM was optimal. Mn(2+) could substitute for Mg(2+), but it was much less efficient than Mg(2+) as the cofactor. The cleavage reaction was stimulated by alkaline pH and KCl at approximately 200 mM. In addition to the high specific activity, Hjc was found to be extremely heat stable. In contrast to the case of SULFOLOBUS:, the Holliday junction resolving activity detected in P. furiosus cell extract thus far is only derived from Hjc.
Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.
Adhesion between desmosomal junctions is mediated by structural proteins of the cadherin family, viz. three desmocollins (DSC) and three desmogleins (DSG). Promoter and primer extension analysis of human DSC3 showed a TATA-less sequence initiating transcription via a cluster of sites upstream of the coding region. Deletion analysis of 1 kb of the promoter showed that expression is regulated between --303 and --203 bp upstream of the start-site of translation. Tertiary structure analysis of this cis-active region (cis 1) revealed a potential DNA 4-way junction which is notably G/C-rich in sequence. PAGE analysis of this region identified four differently migrating forms of the DNA. Structure-specific cleavage of the DNA with bacteriophage T7 endonuclease I showed the slowest migrating form to be either an extended/cruciform or stacked-X 4-way junction. DNA-binding, gel retardation assays of the cis 1 region showed distinct DNA-protein complexes and by competition experiments and using purified junction DNA we show that one of these complexes bound with both sequence and structure specificity to the 4-way junction DNA.
Previously, we demonstrated the interaction of homologous linear duplexes with formation of four-way DNA structures on the model of five PCR products. We propose that homologous duplex interaction is initiated by the nucleation of several dissociated base pairs of the complementary ends of two fragments with Holliday junction formation, in which cross point migration occurs via spooling of DNA strands from one duplex to the other one, finally resulting in complete resolution into new or previously existing duplexes. To confirm that DNA-DNA interaction involves formation of four-way DNA structures with strand exchange at the cross point, we have demonstrated the strand exchange process between identical duplexes using homologous fragments, harboring either biotin label or (32)P-label. Incubation of the mixture resulted in the addition of (32)P-label to biotin-labeled fragments, and the intensity of (32)P-labeling of biotinylated fragments was dependent upon the incubation duration. DNA-DNA interaction is not based on surface-dependent denaturing, as Triton X-100 does not decrease the formation of complexes between DNA duplexes. The equilibrium concentration of Holliday junctions depends on the sequences of the fragment ends and the incubation temperature. The free energy of Holliday junction formation by the fragments with GC and AT ends differed by 0.6 kcal/mol. Electron microscopic analysis demonstrated that the majority of Holliday junctions harbor the cross point within a 300 base pair region of the fragment ends. This insight into the mechanism of homologous duplex interaction extends our understanding of different DNA rearrangements. Understanding of DNA-DNA interaction is of practical use for better interpretation and optimization of PCR-based analyses.
Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.
The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg²⁺ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg²⁺ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.
• FRET
• recombination DNA
• four-way junctions
• fluorescence microscopy
The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg(2+) ions from the oxidised poly(N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg(2+) ions as charge compensating mobile cations. This increase in the Mg(2+) concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg(2+) ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724-4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.
The four-way DNA junction is believed to fold in the presence of metal ions into an X-shaped structure, in which there is pairwise coaxial stacking of helical arms. A restriction enzyme MboII has been used to probe this structure. A junction was constructed containing a recognition site for MboII in one helical arm, positioned such that stacking of arms would result in cleavage in a neighbouring arm. Strong cleavage was observed, at the sites expected on the basis of coaxial stacking. An additional cleavage was seen corresponding to the formation of an alternative stacking isomer, suggesting that the two isomeric forms are in dynamic equilibrium in solution.
The FLP "recombinase" of the 2-micron circle yeast plasmid can resolve synthetic FLP site-Holliday junctions. Mutants of the FLP protein that are blocked in recombination but are normal in substrate cleavage can also mediate resolution. The products of resolution by these mutants are almost exclusively nicked molecules with a protein-bound 3' end. There is no significant asymmetry in strand cleavage (top versus bottom) by the mutants in linear or in circular FLP substrates; nor is there a bias in resolution (toward parentals or toward recombinants) of Holliday junctions (corresponding to top- or to bottom-strand exchange) by wild-type FLP. During normal FLP recombination, a small amount of the expected Holliday intermediate can be detected.
Four-arm Holliday structures are ephemeral intermediates in genetic recombination. We have used an oligodeoxynucleotide system to form immobile DNA junctions, which are stable analogs of Holliday structures. We have probed the equilibrium structure of a junction by means of hydroxyl radicals generated by the reaction of iron(II)EDTA with hydrogen peroxide. The hydroxyl radical cleavage pattern shows twofold symmetry throughout the molecule. Strong protection from hydroxyl radical attack is evident on two strands near the branch site, and weaker protection may be seen four or five residues 3' to the branch site on the other two strands. No other position appears significantly distinct from double-helical DNA controls. From these data, we conclude that the Holliday junction is a twofold symmetric complex whose four arms form two stacking domains.
Lambda site-specific recombination proceeds by a pair of sequential strand exchanges that first generate and then resolve a Holliday junction intermediate. A family of synthetic Holliday junctions with the branch point constrained to the center of the 7 bp overlap region was used to show that resolution of the top strands and resolution of the bottom strands are symmetrical but stereochemically distinct processes. Lambda integrase is sensitive to isomeric structure, preferentially resolving the pair of strands that are crossed in the protein-free Holliday junction. At the branch point of stacked immobile Holliday junctions, the number of purines is preferentially maximized in the crossed (versus continuous) strands if there is an inequality of purines between strands of opposite polarity. This stacking preference was used to anticipate the resolution bias of freely mobile junctions and thereby to reinforce the conclusions with monomobile junctions. The results provide a strong indication that in the complete recombination reaction a restacking of helices occurs between the top and bottom strand exchanges.
In Xer site-specific recombination, two related recombinases, XerC and XerD, mediate the formation of recombinant products using Holliday junction-containing DNA molecules as reaction intermediates. Each recombinase catalyses the exchange of one pair of specific strands. By using synthetic Holliday junction-containing recombination substrates in which two of the four arms are tethered in an antiparallel configuration by a nine thymine oligonucleotide, we show that XerD catalyses efficient strand exchange only when its substrate strands are 'crossed'. XerC also catalyses very efficient strand exchange when its substrate strands are 'crossed', though it also appears to be able to mediate strand exchange when its substrate strands are 'continuous'. By using chemical probes of Holliday junction structure in the presence and absence of bound recombinases, we show that recombinase binding induces unstacking of the bases in the centre of the recombination site, indicating that the junction branch point is positioned there and is distorted as a consequence of recombinase binding.
Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.
We have carried out fluorescence resonance energy transfer (FRET) measurements on four-way DNA junctions in order to analyze the global structure and its dependence on the concentration of several types of ions. A knowledge of the structure and its sensitivity to the solution environment is important for a full understanding of recombination events in DNA. The stereochemical arrangement of the four DNA helices that make up the four-way junction was established by a global comparison of the efficiency of FRET between donor and acceptor molecules attached pairwise in all possible permutations to the 5' termini of the duplex arms of the four-way structure. The conclusions are based upon a comparison between a series of many identical DNA molecules which have been labeled on different positions, rather than a determination of a few absolute distances. Details of the FRET analysis are presented; features of the analysis with particular relevance to DNA structures are emphasized. Three methods were employed to determine the efficiency of FRET: (1) enhancement of the acceptor fluorescence, (2) decrease of the donor quantum yield, and (3) shortening of the donor fluorescence lifetime. The FRET results indicate that the arms of the four-way junction are arranged in an antiparallel stacked X-structure when salt is added to the solution. The ion-related conformational change upon addition of salt to a solution originally at low ionic strength progresses in a continuous noncooperative manner as the ionic strength of the solution increases. The mode of ion interaction at the strand exchange site of the junction is discussed.
The four-way junction between DNA helices is the central intermediate in recombination, and the manner of its interaction with resolvase enzymes can determine the genetic outcome of the process. A knowledge of its structure is a prerequisite to understanding the interaction with proteins, and there has been recent progress. Here we use fluorescence energy transfer to determine the relative distances between the ends of a small DNA junction, and hence the path of the strands. Our results are consistent with the geometry of an 'X'. The interconnected helices are juxtaposed so that the continuous strands of each helix generate an antiparallel alignment, and the two interchanged strands do not cross at the centre. The acute angle of the X structure is defined by a right-handed rotation of the helical axes about the axis perpendicular to the X plane, as viewed from the centre of the X.
An approximate geometry of a stable, four-way DNA junction has been determined in free solution by applying the technique of transient electric birefringence. The current approach consists of (i) construction of a set of six molecules in which two of the four arms of a synthetic junction are elongated by approximately 9-fold (in each of the six possible two-arm combinations), (ii) determination of the ratios of the longest birefringence decay time of each elongated junction to the decay time of a linear control molecule, and (iii) comparison of the experimental ratios with corresponding ratios computed as a function of the junction interarm angle. The result is a set of six angles that define the geometry of the junction. In the presence of magnesium ions, the junction adopts a geometry in which particular pairs of arms approach colinearity. Furthermore, the geometry of the junction is significantly altered in the absence of magnesium, adopting a more uniform structure, although such an effect is not apparent on gels. The application of transient electric birefringence, as described in the current study, should be useful for the characterization of a broad range of tertiary structures in both DNA and RNA.
Cre, the site-specific recombinase from bacteriophage P1, catalyzes a recombination reaction between specific DNA sequences designated as lox sites. The breakage and rejoining of partners during this recombination process must be highly concerted because it has not been possible to detect intermediates of the reaction with wild-type Cre. Several mutant Cre proteins have been isolated that produce significant amounts of a possible intermediate product of the recombination reaction. The product has been identified as a Holliday structure in which one set of the DNA strands of the recombining partners has been exchanged. Wild-type Cre protein is capable of acting on this structure to form recombinant products, which is consistent with this being an intermediate in the recombination reaction. Characterization of the Holliday structure indicated that one set of strands in the recombining partners was always exchanged preferentially before the other set. In addition, it has been found that certain Cre mutants that are unable to carry out recombination in vitro are able to resolve the intermediate. This suggests that these mutants are defective in a step in the reaction that precedes the formation of the Holliday intermediate.
Branched DNA molecules (Holliday structures) are believed to be key intermediates in the process of homologous genetic recombination. However, despite the importance of such structures, their transient nature makes it difficult to analyze their physical properties. In an effort to evaluate several models for the geometry of such branched molecules, a stable, synthetic DNA four-way junction has been constructed. The geometry of the synthetic junction has been probed by gel electrophoresis, utilizing the fact that bent DNA molecules demonstrate reduced mobilities on polyacrylamide gels to an extent that varies with the degree of the bend angle. From the synthetic four-way junction, we have produced a set of molecules in which all combinations of two junction arms have been extended by 105 base-pairs. The electrophoretic mobilities of the extended junctions differ in a manner which indicates that the junction is not a completely flexible structure; nor is it tetrahedral or planar-tetragonal. Instead, the four strands that comprise the DNA four-way junction are structurally non-equivalent. The significance of these observations with regard to previous models for four-way junction geometry is discussed.
General recombination shows a dependence on large regions of homology between the two participating segments of DNA. Many site-specific recombination systems also exhibit a dependence on homology, although in these systems the requirement is limited to a short region (less than 10 base pairs (bp]. We have used the in vitro phage lambda integration reaction to study the role of homology in this model site-specific recombination system. We find that certain non-homologous pairings which are strongly blocked for complete recombination, nevertheless make one pair of strand-exchanges to generate a joint molecule of the Holliday structure type. This result rules out recombination models in which the only homology-dependent step is synapsis (the juxtaposing of the two recombination sites). Our results also reveal a functional asymmetry in the recombination sites. We present models for bacteriophage lambda integrative recombination which accommodate these findings.
A family of novel substrates was designed to enable the efficient accumulation of intermediates in site-specific recombination. Strategically placed nicks allow these "suicide substrates" to initiate the reaction but prevent its completion or reversal. Consequently, it has been possible to determine that lambda site-specific recombination proceeds by a pair of sequential single-strand exchanges. These results rule out that class of models invoking a concerted cutting of all four DNA strands. The sequential strand exchanges are executed in a strictly prescribed order that is the same in both integrative and excisive recombination. This specified order appears to be governed by the arrangement of bound proteins distal to the sites of strand exchange. Furthermore, when provided with an appropriate 5' OH acceptor, the Integrase protein has the capacity to execute a single DNA strand transfer in a nonreciprocal reaction.
The Holliday (four-way) junction is a critical intermediate in homologous genetic recombination. We have studied the structure of a series of four-way junctions, constructed by hybridization of four 80 nucleotide synthetic oligonucleotides. These molecules migrate anomalously slowly in gel electrophoresis. Each arm of any junction could be selectively shortened by cleavage at a unique restriction site, and we have studied the relative gel mobilities of species in which two arms were cleaved. The pattern of fragments observed argues strongly for a structure with two-fold symmetry, based on an X shape, the long arms of which are made from pairwise colinear association of helical arms. The choice of partners is governed by the base sequence at the junction, allowing a potential isomerization between equivalent structural forms. Resolvase enzymes can distinguish between these structures, and the resolution products are determined by the structure adopted, i.e., by the sequence at the junction. In the absence of cations, the helical arms of the junction are fully extended in a square configuration, and unstacking results in junction thymines becoming reactive to osmium tetroxide.
Many site-specific recombinases act by forming and resolving branched Holliday junction intermediates. Previous findings have been consistent with models involving branch migration across the 'overlap region' of obligate homology, located between the staggered sites where the two single-strand exchanges occur. We have investigated the validity of such models in the case of bacteriophage lambda site-specific recombination.
By using synthetic lambda att-site Holliday junctions, incorporating sequence heterologies that impose constraints on branch migration, we have found that the optimal position of the junction for either top-strand or bottom-strand resolution by lambda integrase (Int) is not at the ends, but close to the middle of the seven base-pair overlap region. A minor shift of the branch point around the central base pair caused a remarkable switch in resolution bias. Our findings suggest that branch migration is limited to the central one to three base pairs of the overlap region. They lead to a new model for lambda site-specific recombination, in which there are two symmetrical swaps of two to three nucleotides each, linked by a central isomerization step that causes a change of the stacking interactions between the four junction arms. On the basis of isolated strand-joining reactions carried out by Int in the presence or absence of base complementarity, we propose that sequence homology is sensed during the annealing step prior to strand joining. The new model eliminates mechanistic complications associated with large helical rotations required by branch-migration models.
The results reported here suggest that the recognition of sequence homology in Int-dependent site-specific recombination does not rely primarily on branch migration. The property of cleaving Holliday junctions a few base pairs away from the crossover puts lambda Int into the same category as endonucleases that cleave Holliday junctions in homologous recombination.
A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.
The four-way DNA (Holliday) junction is an important postulated intermediate in the process of genetic recombination. Earlier studies have suggested that the junction exists in two alternative conformations, depending upon the salt concentration present. At high salt concentrations the junction folds into a stacked X structure, while at low salt concentrations the data indicate an extended unstacked conformation. The stereochemical conformation of the four-way DNA junction at low salt (low alkali ion concentration and no alkaline earth ions) was established by comparing the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor molecules attached pairwise in three permutations to the 5' termini of the duplex arms. A new variation of FRET was implemented based upon a systematic variation of the fraction of donor labeled single strands. The FRET results indicate that the structure of the four-way DNA junction at low salt exists as an unstacked, extended, square arrangement of the four duplex arms. The donor titration measurements made in the presence of magnesium ions clearly show the folding of the junction into the X stacked structure. In addition, the FRET efficiency can be measured. The fluorescence anisotropy of the acceptor in the presence of Mg2+ during donor titrations was also measured; the FRET efficiency can be calculated from the anisotropy data and the results are consistent with the folded, stacked X structure.
Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system. Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions. We have constructed an E. coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo. Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products. This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems. The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA. Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid. Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.
Conformational distributions of a four-way DNA junction have been examined by time-resolved fluorescence resonance energy transfer (FRET). A series of dye-labeled junctions were synthesized with donor (fluorescein) and acceptor (tetramethylrhodamine) dyes conjugated to the 5' termini of the duplex arms in all six pairwise combinations. The fluorescence decay of the donor in each junction was measured by time-correlated single-photon counting. The distributions of donor-acceptor (D-A) distances present between each pair of arms were recovered from the donor decays using a continuous Gaussian distribution model. The overall geometry of the four-way junction defined by the six mean D-A distances was consistent with a stacked-X structure, wherein pairs of duplex arms associate to form two continuous domains. Large differences were observed in the widths of the D-A distance distributions, depending on which pair of arms were labeled with the donor and acceptor dyes. Distances measured along the stacking domains were characterized by relatively narrow distributions, indicating that these domains were rigid, whereas distances between stacking domains had broader distributions, reflecting variability in the angle between the two domains. The distances described by broad distributions were overestimated by steady-state FRET measurements. These results suggest that an ensemble of stacked-X structures are present in solution, characterized by differences in the small angle between the stacking domains. Temperature and solvent effects on the recovered distribution widths provide an indication of flexibility in the four-way junction.
Branched DNA molecules provide a challenging set of structural problems. Operationally we define branched DNA species as molecules in which double helical segments are interrupted by abrupt discontinuities, and we draw together a number of different kinds of structure in the class, including helical junctions of different orders, and base bulges (Fig. 1).
The labile protons of two 32-base-pair, four-arm models of immobile Holliday junctions have been studied by two-dimensional 1H nuclear magnetic resonance (NMR) spectroscopy. Overlap of resonances in the imino-imino region of two-dimensional nuclear Overhauser enhancement (NOE) spectra necessitates the use of a multi-pathway approach for obtaining sequence-specific assignments wherein all possible NOE connectivities to the labile protons are utilized, including those from the 2H of adenine, 5CH3 of thymine, and 5H of cytosine. Resonance assignments are obtained for all slowly exchanging imino and cytosine amino protons. Base-pairing up to and including the junction point is found in all four arms of both Holliday junctions. Several cross-arm NOE connectivities are identified and can be used to infer the geometry of the helical stacking domains. The two Holliday junctions studied, which differ only by the exchange of two base pairs at the branch point, appear to have opposite arm stacking geometries. These assignments form an important part of the critical background for detailed NMR analysis of Holliday junction structure and dynamics.
A 32-base-pair model of the Holliday junction (HJ) intermediate in genetic recombination has been prepared and analyzed in-depth by 2D and 3D (1)H NMR spectroscopy. This HJ (J2P1) corresponds to a cyclic permutation of the base pairs at the junction relative to a previously studied HJ [J2; Chen, S.-M., & Chazin, W.J. (1994) Biochemistry 33, 11453-11459], designed to probe the effect of the sequence at the n - 1 position (where n is the residue directly at the branch point) on the stacking geometry. Observation of several interbase nuclear Overhauser effects (NOEs) clearly indicates a strong preference for the isomer opposite that observed for J2, confirming the dependence of stacking isomer preference on the sequence at the junction. As for other model HJs studied, a small equilibrium distribution of the alternate isomer could be identified. A sample of J2P1 was prepared with a single (15)N-labeled thymine residue at the branch point. 1D (15)n-filtered (1)H-detected experiments on this sample at low temperature give strong support for the co-existence of the two stacking isomers and provide a much more direct and accurate measure of the crossover isomer distribution. The comparative analysis of our immobile HJs and a model cruciform structure [Pikkemaat, J.A., van den Elst, H., van Boom, J.H., & Altona, C. (1994) Biochemistry 33, 14896-14907] sheds new light on the issue of the relevance of crossover isomer preference in vivo.
Branched nucleic acid species, three-way and four-way junctions in particular, constitute important structural elements in RNA and DNA, either as part of the architecture (mainly RNA) or as potential intermediates in biological processes (mainly DNA). The number of research reports on branched nucleic acids is growing rapidly. Thus far, comparison of tertiary structures reported by various groups remains difficult as it is hampered through lack of uniform depiction and notation. We therefore propose a set of simple rules that allow a unique classification as well as a uniform depiction of virtually any junction in terms of the base-pairs at the branch point. In its simplest form the methodology describes and arranges the n base-pairs in an n-way (nH) junction but it is easily extended in order to include single-stranded regions, mismatches and penultimate base-pairs. Counting only base-pairs at the branch point, 64 different bulged 3HS2 three-way junctions can be constructed, arranged into eight different classes. For reasons of symmetry, only 36 different immobilized 4H junctions are possible, arranged into six classes, and only 24 tight 3H junctions, in four classes. The number of different junctions in the general nHSm case is 4n, multiplied by 4m when the Sm unpaired bases are included in the calculation.
The Holliday junction is a key intermediate in genetic recombination. This is a four-stranded branched DNA structure, whose double-helical arms are stacked in two domains; two of the strands are roughly helical, and the other two cross over between domains. Switching the strands between these two roles is known as crossover isomerization; this postulated reversal is thought to be one of the key transformations that the Holliday junction can undergo, because it can lead to changing the products from patch to splice recombinants. We present direct evidence that this reaction can indeed occur in Holliday junctions in solution. We have constructed a double-crossover molecule containing a branched junction, constrained not to be in its favored conformation. This junction is released from the double-crossover molecule by digestion with restriction endonucleases. We demonstrate by means of hydroxyl radical autofootprinting that the junction changes its crossover isomer spontaneously when released from the double crossover. We control for the possibility that the experimental protocol causes the isomerization. We also exclude dissociation and interaction with other molecules in solution as contributing to the phenomenon. Thus, crossover isomerization is an authentic spontaneous transformation of Holliday junctions.
The junction-resolving enzymes are a class of nucleases that introduce paired cleavages into four-way DNA junctions. They are important in DNA recombination and repair, and are found throughout nature, from eubacteria and their bacteriophages through to higher eukaryotes and their viruses. These enzymes exhibit structure-selective binding to DNA junctions; although cleavage may be more or less sequence-dependent, binding affinity is purely related to the branched structure of the DNA. Binding and cleavage events can be separated for a number of the enzymes by mutagenesis, and mutant proteins that are defective in cleavage while retaining normal junction-selective binding have been isolated. Critical acidic residues have been identified in several resolving enzymes, suggesting a role in the coordination of metal ions that probably deliver the hydrolytic water molecule. The resolving enzymes all bind to junctions in dimeric form, and the subunits introduce independent cleavages within the lifetime of the enzyme-junction complex to ensure resolution of the four-way junction. In addition to recognising the structure of the junction, recent data from four different junction-resolving enzymes indicate that they also manipulate the global structure. In some cases this results in severe distortion of the folded structure of the junction. Understanding the recognition and manipulation of DNA structure by these enzymes is a fascinating challenge in molecular recognition.
- G Carlstrom
- W J Chazin
Carlstrom, G. & Chazin, W. J. (1996) Biochemistry 35, 3534–
3544.
- S E Nunes-Düby
- L Matsomoto
- A Landy
Nunes-Düby, S. E., Matsomoto, L. & Landy, A. (1987) Cell 50,
779–788.
- R M Clegg
- A I H Murchie
- A Zechel
- D M J Lilley
Clegg, R. M., Murchie, A. I. H., Zechel, A. & Lilley, D. M. J.
(1994) Biophys. J. 66, 99–109.
- J A Pikkemaat
- H Van Den Elst
- J H Van Boom
- C Altona
Pikkemaat, J. A., van den Elst, H., van Boom, J. H. & Altona, C.
(1994) Biochemistry 33, 14896–14907.
- C Altona
Altona, C. (1996) J. Mol. Biol. 263, 568–581.
- S M Chen
- F Heffron
- W J Chazin
Chen, S. M., Heffron, F. & Chazin, W. J. (1993) Biochemistry 32,
319–326.
- P S Eis
- D P Millar
Eis, P. S. & Millar, D. P. (1993) Biochemistry 32, 13852–13860.
- X Li
- H Wang
- N C Seeman
Li, X., Wang, H. & Seeman, N. C. (1997) Biochemistry 36,
4240–4247.
- J P Cooper
- P J Hagerman
Cooper, J. P. & Hagerman, P. J. (1987) J. Mol. Biol. 198, 711–719.
- R Holliday
Holliday, R. (1964) Genet. Res 5, 282–304.