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N C Seeman

Publications and source records attributed to N C Seeman.

At least 37 records · Page 2Linked to original sources
PubMed1996

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Base Sequence↗
PubMed1996

The design and engineering of nucleic acid nanoscale assemblies.

It is possible to design DNA molecules that can form unusual structures and topologies. Stable DNA-branched junctions have been used to construct polyhedral catenated molecules with the connectivities of a cube and of a truncated octahedron. The truncated octahedron has been constructed following a solid-support-based methodology. Branched-DNA molecules are flexible, suggesting that triangular and deltahedral DNA objects should be favored as the components of two- and three-dimensional nucleic acid arrays. DNA polyhedra are complex catenanes. The engineering of single-stranded DNA knots and catenanes exploits the fact that a node can be equated with a half-turn of DNA.

Base Sequence↗
PubMed1995

Structural domains of DNA mesojunctions.

Holliday junctions are central intermediates in the process of genetic recombination. These DNA molecules contain four double helices that flank a central branch point, so that each of the four strands that constitute the junction is associated with two different helices. Previously, we have shown that such branched junctions are a special subset of the class of DNA molecules containing multiple strands, in which each strand is associated with two double helices [Du, S. M., Zhang, S., & Seeman, N. C. (1992) Biochemistry 31, 10955-10963]. Conventional branched molecules, such as the Holliday junction, contain helices whose axes can all be drawn to radiate from a central point. The other molecules of the class contain helices whose axes are circumferential about the central point. If all of the helices are circumferential, the molecule is called an antijunction; if the molecule contains a mixture of radial and circumferential helices, it is called a mesojunction. The properties of the molecule are a function of the even or odd nature of the number of helical half-turns in the circumferential helices. Previously, a four-strand antijunction and three-strand and four-strand mesojunctions containing three half-turns (16 nucleotide pairs) per helix were constructed and characterized. Here, we have attempted to construct the analogous molecules containing two half-turns (10 nucleotide pairs) in each helix. The three-strand mesojunction and the four-strand antijunction are not stable under conditions suitable for analysis. We have characterized the two four-strand mesojunctions that are stable by Ferguson analysis, thermal denaturation, and Fe(II)EDTA2- autofootprinting. In addition, we have characterized a four-strand mesojunction containing 14 nucleotide pairs in its circumferential helices and have shown that its favored conformer is different from that of a closely related molecule containing 16 nucleotide pairs in its circumferential helices.

Base Sequence↗
PubMed1995

Topological transformations of synthetic DNA knots.

Two synthetic DNA molecules that can be knotted have been employed as substrates for E. coli DNA topoisomerases I and III. Both molecules contain 104 nucleotides, including sequences that can form two single-turn helical domains, connected by single-stranded oligo(dT) linkers in an X-Y-X'-Y' pairing motif. One of the knots can be ligated to form cyclic molecules with the topologies of a circle, a trefoil knot with negative nodes, or a figure-8 knot. Cyclic molecules constructed from the other molecule can form a circle, a figure-8 knot, and trefoil knots with either positive or negative nodes. The topologically negative nodes in these knots are derived from right-handed B-DNA, and the positive nodes are derived from left-handed Z-DNA. The topoisomerases can catalyze the interconversion of the different topological forms of these molecules, as a function of solution conditions and the extent to which they favor B-DNA or Z-DNA. The enzymes appear to catalyze a single strand-passage event at a time. The topoisomerases can catalyze strand passage events involving both positive and negative nodes as substrates. Gel retention experiments show that both knots can bind up to four molecules of E. coli DNA topoisomerase I. The thermal denaturation of the domains of a trefoil knot closely related to these knots suggests that the two helical domains are uncoupled, so the single-stranded linkers in the knots are not taut. Chemical ligation experiments yield a distribution of products similar to those of enzymatic ligation, showing that the ATP cofactor in DNA knot ligation does not appear to skew the products markedly. Knots that are stressed by being placed in unfavorable solution conditions have been shown to be a highly sensitive system for detecting topoisomerase activity.

Adenosine Triphosphate↗
PubMed1994

Symmetric Holliday junction crossover isomers.

The Holliday junction is a four-stranded branched DNA structure found as an intermediate in genetic recombination. Naturally occurring Holliday junctions have homologous (2-fold) sequence symmetry; this symmetry renders the site of the branch unstable, because the molecules can undergo an isomerization called branch migration. For the past decade, these molecules have been modeled in non-migrating "immobile" branched junctions lacking this symmetry. The solution structure derived from immobile branched junctions is a two-domain DNA structure, in which a particular pair of strands forms the crossover between domains, and the other pair of strands has a structure similar to that seen in DNA double helices; reversal of these roles has not been apparent. We investigate here whether this bias ("crossover preference") for particular strands to be the crossover pair extends to structures that contain symmetric sequences flanking the branch point. We employ nicked symmetric immobile junctions in a competition assay to determine the extent of crossover preference. We have measured all six of the 2-fold symmetric dinucleotide sequences that can flank a branch point, and find small preferences (< 650 cal/mol) at 4 degrees C. The largest preference decreases when the 2-fold symmetry is extended to tetranucleotides. The system contains an internal control on systematic errors, because there are 4-fold symmetric dinucleotide sequences that should show no bias; the preferences we measure for them are below or similar to our estimated errors. We have calibrated our experiment by measuring the crossover preferences for a previously characterized immobile junction; the preference is slightly larger than the largest preference seen for a symmetric crossover. We conclude that large crossover preferences are not characteristic of junctions with extensive symmetry, and are thus unlikely to have important consequences for genetic recombination.

Base Sequence↗
PubMed1994

Cleavage of double-crossover molecules by T4 endonuclease VII.

DNA double-crossover molecules containing two Holliday junctions have been prepared and treated with endonuclease VII, the resolvase from bacteriophage T4. One molecule contains antiparallel double-helical domains, and the other molecule contains parallel domains. The parallel double-crossover model system has been made tractable by closing the free ends of the molecule, to convert it to a catenane. The products resulting from the two substrates differ substantially. The molecule containing antiparallel helical domains is cleaved three nucleotides 3' to the crossover points, in a fashion similar to single Holliday junction analogs. The molecule containing parallel helical domains is cleaved, but the major points of scission are five nucleotides 5' to a branch point on the crossover strands and six nucleotides 3' to the same branch point on the non-crossover strands. The major sites of scission reflect features of molecular symmetry in each case, suggesting that the resolvase recognizes structural features. The cleavage results suggest that the antiparallel structure is the natural substrate, if the Holliday junction is unconstrained within the cell. It is straightforward to reconcile antiparallel Holliday junctions with the conventional parallel paradigm of recombination. Nevertheless, the cleavage of the parallel molecule shows that a parallel substrate could also be cleaved symmetrically by endonuclease VII (but with different products) if the molecule were constrained to assume that conformation within the cell.

Base Sequence↗
PubMed1994

Holliday junction crossover topology.

The Holliday junction is a key intermediate in genetic recombination. It consists of four strands of DNA that associate to form four double-helical arms. Studies over the past decade with asymmetric analogs of Holliday junctions have shown that the four arms stack to generate two stacking domains. This arrangement of arms results in two strands with a roughly helical structure, and two that contain a crossover structure connecting the domains. Both parallel and antiparallel orientations of the helical strands are possible, although the antiparallel orientation is favored. In principle, there are two possible isomers of the Holliday junction, depending on which pairs of strands contain the crossover structure; isomerization between these two structures is key to many molecular models of recombination. Isomerization of parallel domain structures entails large end-to-end helical rotations if braiding of the crossover strands is to be avoided. We have examined the ability of the crossover strands to braid. This has been done by employing a double crossover molecule, whose termini are all hairpin loops. Such a molecule is a catenane of two single strands of DNA, whose linking number is a function of the sign of the node at the crossover. We have prepared linking standards by means of topological protection techniques, and have compared them to the catenane formed by the double crossover molecule. We find no evidence for braiding of the strands. Furthermore, no braided structures can be detected when the double crossover molecule is treated with Escherichia coli DNA topoisomerase I in the presence of varying amounts of Mg2+ cations.

Base Sequence↗
PubMed1994

The construction of a trefoil knot from a DNA branched junction motif.

A DNA trefoil (3(1)) knot has been constructed from a 104-nucleotide molecule whose strands form a 3-arm branched junction motif. This construction tests the notion that a node in a DNA knot can be equated with a half-turn of double-helical DNA, and is consistent with that concept. Of five 104-mer sequences tested, only one produces high yields of the target knot. The other molecules produce larger quantities of circular material and of a knot containing more nodes. The key features that differentiate the successful design from the others are (1) the ligation takes place in the linker region between helical domains and (2) only six nucleotide pairs are used for each of the double-helical arms of the junction. The successful design separates the double-helical regions from each other by a spacer containing two deoxythymidine nucleotides at the site of the branched junction.

Base Sequence↗
PubMed1993

Symmetric immobile DNA branched junctions.

Branch migration is an isomerization of Holliday recombination intermediates that arises from their homologous (2-fold) sequence symmetry. This isomerization relocates the branch point in an apparently random fashion and thereby complicates the study of the physical and structural properties of these structures. For the past decade, these properties have been studied in low-symmetry immobile junctions, whose sequence asymmetry eliminates branch migration. The asymmetric findings of many of these studies suggest the need for a system combining both immobility and symmetry. Double-crossover DNA molecules have been used to create molecules with both these properties. Immobility is achieved by flanking one crossover with a symmetric junction and the other crossover with an asymmetric junction. Close torsional coupling between the two junctions renders the symmetric junction immobile. These molecules will enable the characterization of thermodynamic, structural, dynamic, liganding, and substrate properties of symmetric branched DNA molecules in a sequence-specific fashion.

Base Sequence↗
PubMed1993

DNA double-crossover molecules.

DNA molecules containing two crossover sites between helical domains have been suggested as intermediates in recombination processes involving double-strand breaks. We have modeled these double-crossover structures in an oligonucleotide system. Whereas the relative orientations of the helical domains must be specified in designing these molecules, there are two broad classes of the molecules, the parallel, DP, and antiparallel, DA, molecules. The distance between crossover points must be specified as multiples of half-turns, in order to avoid torsional stress in this system; hence, there are two further subdivisions, those double-crossover molecules separated by odd, O, and even, E, numbers of half-turns. In addition, the parallel molecules with odd numbers of half-turns between crossovers must be divided into those with an excess major or wide-groove separation, W, or those with an excess minor- or narrow-groove separation, N. We have constructed models of all five of these classes, DAE, DAO, DPE, DPOW, and DPON. DPE molecules containing 1 and 2 helical turns between crossovers have been constructed; the DAE molecule contains 1 turn between crossovers, and the DAO, DPOW, and DPON molecules contain 1.5 helical turns between crossovers. None of the parallel molecules is well-behaved; the molecules either dissociate or form multimers when visualized on native polyacrylamide gels. In contrast, antiparallel molecules form single bands when assayed in this fashion. Hydroxyl radical autofootprinting analysis of these molecules reveals protection at expected sites of crossover and of occlusion, suggesting that all the complexes contain linear helix axes that are roughly coplanar between crossovers. However, the DPOW molecule and the DPE molecule with 2 turns between crossovers show decreased protection in the portion between crossovers, suggesting that their helices may bow in response to charge repulsion. We conclude that the helices between parallel double crossovers must be shielded from each other or distorted from linearity if they are to participate in recombination. We have analyzed the possibilities of branch migration and crossover isomerization in double-crossover molecules. Parallel molecules need no sequence symmetry beyond homology to branch migrate, but the sequence symmetry requirements for antiparallel molecules restrict migration to directly repetitive segments that iterate the sequence between crossovers. Crossover isomerization appears to be a very complex process in parallel double-crossover molecules, suggesting that it may be catalyzed by topoisomerases if it occurs within the cell.

Base Sequence↗
PubMed1993

Tight single-stranded DNA knots.

Trefoil (3(1)) and figure-8 (4(1)) knots have been synthesized from DNA molecules containing two single-turn helical domains, linked by four oligodeoxythymidine linkers. Both topologies are derived from the same DNA molecule. The tightest knots are fashioned by minimizing the lengths of the linkers. The shortest equal-length linkers from which a trefoil knot can be made readily are seven nucleotides long, in a 74-nucleotide molecule, whereas those in the shortest figure-8 knot are six nucleotides long, in a 70-nucleotide molecule. In addition to these limiting knots, other knots containing 80, 88, 96 and 104 nucleotides have been constructed. The mobilities of these molecules on denaturing gels show the conventional logarithmic dependence on length. Ferguson analysis of their mobilities indicates a linear dependence of surface area on length. The 80-mer trefoil knot is the tightest molecule that can be restricted in both domains.

Base Sequence↗
PubMed1993

A scanning tunnelling microscopy study of the formation and chemical activation of step defects on the basal plane of pyrolytic graphite.

Scanning tunnelling microscopy is used to monitor etching of the basal plane of highly orientated pyrolytic graphite by ozone, oxygen and nitric acid. These treatments are seen to produce numerous single and multilayer step defects. Subsequent modification of the graphite sheet edges flanking these cavities by cyanuric chloride, TiCl4 and other reagents is shown to activate the edges, thereby making them capable of covalently binding various molecules.

Acrylates↗
PubMed1993

New methods for depositing and imaging molecules in scanning tunneling microscopy.

Methods and apparatus are described to deposit and image molecules by scanning tunneling microscopy (STM) under an inert atmosphere. Three methods of applying molecules have been evaluated: equilibrium adsorption from the vapor phase, sublimation, and electrospraying. Using these methods, a variety of organic and biopolymer molecules have been deposited and imaged on graphite and on gold (111), grown epitaxially on mica. Compared with alternatives, such as the use of high vacuum apparatus or glove boxes, these procedures offer some important advantages: they are inexpensive, convenient, and more rapid. Mercaptoethanol, ethanolamine, ethanol, acetic acid, and water produce two-dimensional crystalline adlayers on gold substrates, when they are introduced into the scanning cell as vapors. These adlayers are assumed to involve hydrogen bonding of the molecules to an oxide of gold formed on the surface. Electrospraying protein solutions on gold surfaces yielded images of individual protein molecules with lateral dimensions close to those measured by X-ray analysis, and thicknesses of 0.6-1.3 nm. In the case of metallothionein, the known internal domain structure of the molecule was reproducibly observed. No detailed internal structure could be resolved in the other examples examined.

Acetates↗
PubMed1992

DNA junctions, antijunctions, and mesojunctions.

Antijunctions and mesojunctions are new classes of multistranded DNA complexes. They represent a generalization of DNA branched junction complexes, such as the Holliday recombination intermediate. Each strand of a conventional branched junction participates in two different double helices, and this is also true for mesojunctions and antijunctions. The helix axes of conventional branched junction complexes may be drawn to converge at a point, but this convergence occurs for lines drawn perpendicular to the helix axes of antijunctions. Mesojunctions are complexes that mix these features of junctions and antijunctions. Antijunction complexes require an even number of strands. We have synthesized the mesojunction containing three strands, the two mesojunctions containing four strands, and the antijunction containing four strands; we compare them with branched junctions containing three and four strands, derived by permutations of the same sequences. Each double helix is designed to contain 1.5 turns of DNA. A tendency to oligomerize makes it difficult to capture antijunctions and mesojunctions in stable discrete complexes, in contrast to conventional branched junctions. For both three-strand and four-strand complexes, Tm is highest for conventional branched junctions. Ferguson analysis indicates similarities in the occluded surface area of junctions, antijunctions, and one four-strand mesojunction, but the other four-strand mesojunction has a much lower apparent surface area. Hydroxyl radical cleavage patterns suggest that the four-strand antijunction and the low-surface-area four-strand mesojunction form stacking domains, analogous to the behavior of conventional branched junctions. These new structures are related to replicational and recombinational intermediates and to single-stranded nucleic acid knots.

Base Sequence↗
PubMed1992

Thermodynamics of DNA branching.

Branched DNA molecules arise transiently as intermediates in genetic recombination or on extrusion of cruciforms from covalent circular DNA duplexes that contain palindromic sequences. The free energy of these structures relative to normal DNA duplexes is of interest both physically and biologically. Oligonucleotide complexes that can form stable branched structures, DNA junctions, have made it possible to model normally unstable branched states of DNA such as Holliday recombinational intermediates. We present here an evaluation of the free energy of creating four-arm branch points in duplex DNA, using a system of two complementary junctions and four DNA duplexes formed from different combinations of the same set of eight 16-mer strands. The thermodynamics of formation of each branched structure from the matching pair of intact duplexes have been estimated in two experiments. In the first, labeled strands are allowed to partition between duplexes and junctions in a competition assay on polyacrylamide gels. In the second, the heats of forming branched or linear molecules from the component strands have been determined by titration microcalorimetry at several temperatures. Taken together these measurements allow us to determine the standard thermodynamic parameters for the process of creating a branch in an otherwise normal DNA duplex. The free energy for reacting two 16-mer duplexes to yield a four-arm junction in which the branch site is incapable of migrating is + 1.1 (+/- 0.4) kcal mol-1 (at 18 degrees C, 10 mM-Mg2+). Analysis of the distribution of duplex and tetramer products by electrophoresis confirms that the free energy difference between the four duplexes and two junctions is small at this temperature. The associated enthalpy change at 18 degrees C is +27.1 (+/- 1.3) kcal mol-1, while the entropy is +89 (+/- 30) cal K-1 mol-1. The free energy for branching is temperature dependent, with a large unfavorable enthalpy change compensated by a favorable entropy term. Since forming one four-stranded complex from two duplexes should be an entropically unfavorable process, branch formation is likely to be accompanied by significant changes in hydration and ion binding. A significant apparent delta Cp is also observed for the formation of one mole of junction, +0.97 (+/-0.05) kcal deg-1 mol-1.

Base Composition↗