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Biomedical subjects

N C Seeman

Publications and source records attributed to N C Seeman.

At least 73 records · Page 4Linked to original sources
PubMed1988

A Holliday recombination intermediate is twofold symmetric.

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.

Free Radicals↗
PubMed1988

T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs.

We have formed four-arm branched DNA junctions that contain no more than a single base pair of branch migratory freedom. Recently, we have shown that these Holliday junction analogs have twofold symmetric protection patterns in solution when probed with hydroxyl radicals: two opposite strands of one junction show extensive protection near the branch point, while the other pair of opposite strands is virtually as susceptible as a double helix. In a different junction, the hydroxyl radical protection pattern is reversed. These patterns suggest that a crossover-isomer bias exists in these molecules and that the protected strands form the crossover between helices. Here, we examine the cleavage pattern of these structures when they are resolved by T4 endonuclease VII. Junctions are formed from a single shamrock-shaped molecule, which contains 5', 3', or internal labels. The enzyme shows a preference for resolving these modified junctions at sites near those protected from hydroxyl radicals. This result suggests that only crossover strands in a Holliday junction are cleaved, and thus an odd number of crossover isomerizations must occur when flanking markers are exchanged.

Base Sequence↗
PubMed1988

Physical models for exploring DNA topology.

Two types of physical models have been developed for treating DNA molecules whose topology is of interest. The two model motifs combine jacks-and-straws molecular representations with flexible tubing in different proportions. Both motifs present a low-resolution construct of DNA that retains helix axes, strand individuality and the distinguishability of the major and minor grooves. Molecules whose double helix axes are branched are modelled by stiff double helices and flexible branch sites. Supercoiled and knotted DNA molecules are modelled on a smaller scale, in a system in which a flexible backbone is supported by a series of stiff helical struts; removal of this scaffolding immediately reveals the linking of the strands. The models are light and easy to construct. They may be used either for demonstrations or as a research tool that assists the interpretation data.

Audiovisual Aids↗
PubMed1987

Simulation of double-stranded branch point migration.

A structural and dynamic model has been developed for the branch point formed when two DNA double helices exchange strands during genetic recombination. This model, which generalizes most previous structural models, maintains the twofold symmetry inherent in the covalent and hydrogen bonded structure, yet has three degrees of freedom about virtual bonds, constituting a simplified junction. Using this structural model, a three-step dynamic model for branch point migration has been developed: longitudinal diffusion about the virtual bonds to achieve a structure in which the helix axes are approximately parallel; opening of the base pairs; and rotary diffusion about the helix axis to effect a migratory event. The model, which includes the possible role of electrostatic interactions, solves problems inherent in previous treatments. We find that no significant electrostatic torques arise that promote branch point migration. The absence of a kinetic mechanism to circumvent thermodynamic barriers due to mispairing suggests that an energy source is used for those situations in living systems.

Base Composition↗
PubMed1986

Three-arm nucleic acid junctions are flexible.

Nucleic acid junctions are stable analogs of branched DNA structures which occur transiently in living systems. We show here that junctions which contain three double helical arms can be enzymatically oligomerized, using conventional sticky-ended ligation procedures, to create larger complexes. The products consist of a series of linked junctions separated by 20 base pairs. Junction dimers are formed that have free termini only, whereas trimers and larger species are found to be both unclosed and cyclized. The formation of a series of macrocyclic products which, surprisingly, begins with trimers and tetramers indicates that this junction is flexible about a bending axis, and perhaps twist-wise as well. We have obtained the same results from three different 3-arm junctions, two in which the junction is flanked by a 3 Watson-Crick base pairs, and one in which a G-G base pair flanks the junction.

Base Composition↗
PubMed1985

NMR analysis of DNA junctions: imino proton NMR studies of individual arms and intact junction.

The NMR resonances from the hydrogen-bonded guanine and thymine imino protons of base pairs in the four separate complexes forming the arms of a stable DNA four-arm junction have been assigned by using sequential nuclear Overhauser effects connecting protons in adjacent pairs. Comparison of the spectra of these individual duplex arms with that of the intact four-stranded junction suggests that base pairing occurs at the site of branching. The presence of new resonances in the spectrum of the junction can be inferred from comparison of the junction spectrum with the simulated spectra of the four individual arms. In addition, upfield shifts of the ring protons in the base pairs at the penultimate positions in the complex are observed, consistent with a change in the structure at the site of branching. These studies represent the first stage of a detailed analysis of the structure and dynamics of a DNA junction.

Base Sequence↗
PubMed1985

Macromolecular design, nucleic acid junctions, and crystal formation.

The simplest form of macromolecular design involves the ligation of nucleic acids. Recent results on the concatenation of nucleic acid junctions show that these molecules can act as fairly rigid macromolecular valence clusters on the nanometer scale. These clusters can be joined to form closed stick figures in which each edge is double helical DNA or RNA and each vertex is a nucleic acid junction. The geometrical criteria for forming discrete-closed and periodic structures from these components are established. The helicity of each edge limits the possible structures that can be formed. The formation of a periodic array from nucleic acid junction building blocks is compared with the crystallization of molecular systems. This comparison leads to a new interpretation of the nature of order in the solid state for molecular crystals. The suggestion is made that the structure of a solid molecular system described by the fewest unique orthogonal (Fourier) components is the one which will be entropically favored, since it contains the least information. This is the crystalline state, with a small number of molecules per asymmetric unit. The free energy from the proposed entropic driving force responsible for this behavior is available, in principle, to correct small deviations from ideality in forming covalent crystals from nucleic acid junction components, as well as in non-bonded molecular systems. Nucleic acid junction periodic arrays provide an appropriate vehicle with which to test this interpretation.

Base Sequence↗
PubMed1985

Physical characterization of a nucleic acid junction.

Normally unstable transient states of DNA, in which the linear duplex branches to form junctions with three or more arms, can be studied at the oligonucleotide level if their sequences are carefully selected. We have designed a series of oligonucleotide complexes with sequences that are restricted to prevent any major overlap among the arms, and chosen to exhibit high equilibrium stabilities, as well. The electrophoretic mobility of these complexes on polyacrylamide gels permits us to demonstrate formation of a stable four-strand complex with 1:1:1:1 stoichiometry. We review here the evidence for formation of a stable stoichiometric junction, and present new circular dichroism data showing that the arms remain in B helix geometry within the complex, and that no significant loss of structure occurs on forming a junction.

Circular Dichroism↗
PubMed1983

Design of immobile nucleic acid junctions.

Nucleic acids that interact to generate structures in which three or more double helices emanate from a single point are said to form a junction. Such structures arise naturally as intermediates in DNA replication and recombination. It has been proposed that stable junctions can be created by synthesizing sets of oligonucleotides of defined sequence that can associate by maximizing Watson-Crick complementarity (Seeman N. C., 1981, Biomolecular Stereodynamics. Adenine Press, New York. 1: 269-278; Seeman, N. C., 1982, J. Theor. Biol. 99:237-247.) To make it possible to design molecules that will form junctions of specific architecture, we present here an efficient algorithm for generating nucleic acid sequences that optimize two fundamental properties: fidelity and stability. Fidelity refers to the relative probability of forming the junction complex relative to all alternative paired structures. Calculations are described that permit approximate prediction of the melting curves for junction complexes.

DNA↗
PubMed1983

Fourth rank immobile nucleic acid junctions.

An immobile nucleic acid junction composed of four dodecanucleotides has been designed according to principles of minimum symmetry aided by equilibrium calculations, and has been synthesized by automated phosphotriester techniques. We can demonstrate its tetrameric character and its 1:1:1:1 stoichiometry by gel electrophoresis. Thermal denaturation monitored by ultraviolet hyperchromism indicates that the complex is stable relative to its component arms. High resolution NMR spectroscopy suggests that this junction exists in more than one conformer at room temperature. The data from this junction are compared with the data from a similar junction composed of four hexadecanucleotides.

Base Sequence↗