DNA junctions

FIG. 13 Synthetic DN A motifs for the construction of DNA framework Three- [75] (13) and four-arm (14) DNA junction [8] DNA double-crossover (DX) molecules 15,16 were used for initial studies of enzymatic oligomerization [79]. The DX motif 17, containing four cohesive ends of individual nucleotide sequence, was used for the construction of two-dimensional DNA crystals [80]. 

FIG. 14 Construction of periodic framework and geometric objects from DNA. (a) Construction of two-dimensional DNA lattices from tetravalent four-arm DNA junctions (14) [8]. (b) Synthesis of a macrocyclic molecule from bivalent three-arm DNA junctions (13a) containing two cohesive ends [83]. For simplification, linear double-helical stretches are represented by parallel lines. 

Stros, M. and Muselikova, E. (2000) A role of basic residues and the putative intercalating phenylalanine of the HMGBl-box B in DNA supercoiling and binding to four-way DNA junctions. J. Biol. Chem. 275, 35699-35707. 

The three-way DNA junction (also called Holliday junction) has been studied by single molecule AFM analysis [48]. DNA molecules have been dispersed in saline buffer injected in a fluid cell mounted on the top of the APTES mica. Fig. 10 presents two consecutive AFM scans (separated by approximately 3 min) of 301 bp DNA molecules containing a 7 bp hairpin. Also Fig. 11 presents the AFM images of representative slipped strand DNA structures. The 3D projections of the (CGGjso (CCGjso slipped strand structures... 

Comparative gel electrophoresis was first apphed to the four-way (Holliday) DNA junction. This revealed the stacked X-structure of the junction, its dependence on the presence of metal ions, and the occurrence of two alternative conformers of the structure (Duckett el al., 1988). The stacked X-structure was confirmed by crystallography a decade after the electrophoretic experiments (Eichman el al., 2000 Ortiz-Lombardia et al., 1999). Since then, comparative gel electrophoresis has scored many more successes, with no failures to date. This has been reviewed recendy (Lilley,... 

In principle the global structure of an RNA junction could be determined when it is complexed with bound protein. This has not been accomplished to date in RNA, but it has proven itself for DNA junctions. For example, the structure of DNA junctions bound by the junctionresolving enzymes T4 endonuclease VII (Pohler et al., 1996) and T7 endonuclease I (Declais et al., 2003) have both been determined by comparative gel electrophoresis. It was found that both proteins substantially alter the global shape of the DNA junction in different ways. These structures were both recendy confirmed by X-ray crystallography (Biertiimpfel et al., 2007 Hadden et al., 2007), showing that comparative gel electrophoresis functions reliably for protein complexes. There is no reason to expect that the method would not work equally well for RNA junction complexes. The binding of the ribosomal protein SI 5 to a three-way RNA junction has been studied by an electrophoretic approach that is related to comparative gel electrophoresis (Batey and Williamson, 1998). 

In a recent development, FRET has been used to study the dynamics of single DNA junctions while under the application of stretching force (Hohng et al, 2007) this experiment should be directly applicable to branched RNA species. 

Murchie, A. I. H., Clegg, R. M., von Kitzing, E., Duckett, D. R., Diekmann, S., and Lilley, D. M. J. (1989). Fluorescence energy transfer shows that the four-way DNA junction is a right-handed cross of antiparallel molecules. Nature 341, 763—766. 

Since the preferential interaction coefficient T can be interpreted in terms of Donnan equilibrium [66, 74, 96, 97], a grand canonical Monte Carlo (GCMC) simulations could be used to determine it, from a knowledge of the slope of salt concentration c3 as a function of the polyion concentration cD [68, 73, 74]. Such an analysis was carried out by Olmsted and Hagerman for a tetrahedral four-arm DNA junction, based on the so-called primitive model of the electrolyte [74]. 

Fig. 5. Protein-RNA fusion. Covalent RNA-protein complexes can be generated by ligation of a DNA-puromycin linker to the in vitro transcribed mRNA. During in vitro translation, the ribosome stalls at the RNA-DNA junction. Puromycin can then bind to the ribosomal A-site. The nascent polypeptide is thereby transferred to puromycin. The resulting covalendy linked complex of mRNA, puromycin, and peptide can be used for selection experiments. After affinity selection, the bound complexes are eluted and subsequently the mRNA is amplified by RT-PCR.

Fig. 8 Schematic representation of DNA junctions and crossover tiles. Motif 1 is a branched DNA junction with three arms and motif 2 with four arms. Every terminal in the arm is an unpaired ssDNA. The ssDNA acts as sticky ends , which may pair with another complementary strand. The two motifs 3 and 4 are two different antiparallel double-crossover molecules containing an even number of half-helical turns between branch points (DAE) or an odd number (DAO). They are more stable and thus usually applied. Oligonucleotide strands are individually represented with different colors...

Fig. 9 Assembly of DNA junctions, a Four of the junctions in motif 2 are complexed to yield the structure in motif 5. The complex has maintained open valences so that it could be extended by the addition of more monomers, b Square lattice formed from four-arm junctions held in a square-planar configuration (6) by protein RuvA, with TEM image of the lattice shown beneath. The scale bar represents 100 nm. Reprinted with permission from [47], c ID self-assembly of the motif 7 derives into a railroad track-like array 8, and the 2D self-assembly produces a lattice array 9. An AFM image of array 9 is shown beneath with a scan size of 400 x 400 nm2. Adapted with permission from [45,46]...

Single-molecule FRET has been used to study the kinetics of unfolding of the human telomeric intramolecular G-quadruplex, the DNA-binding orientation of an E.coli REP monomer to a ss/ds DNA junction, four-way... 

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

XPG is a structure-specific endonuclease with two discernable roles in NER. It makes the incision 3 to the lesion and has nuclease activity on substrates with single/double-stranded DNA junctions mimicking the open intermediate in the NER reaction [83, 84], Prior to exerting its catalytic activity, it plays a structural role in assembling the preincision complex in NER. The presence, but not the catalytic activity of XPG is required for the assembly of the preincision complex [85, 86],... 

Figure 18. Extended hybrid inorganic-nucleic acid structures based on inorganic DNA junctions (adapted from References 168,169, and 172).

Extended structures can be rationally built by appropriate combination of the metal-based DNA junctions with nucleic acid entities that have a specific degree of complementarity. Hybridization of two four-arm, [Ni(cyclam)] -based DNA junctions that had complementary DNA arms showed results consistent with the formation of high-order, infinite structures [Fig. 18(a)] (168). Mixing of complementary three-arm, [Fe(bpy)3] -based DNA junctions led to mesoscopic structures (171). The combination of three two-arm, [Fe(tpy)2] " -based DNA junctions that had arms intermolecularly, pairwise complementary led to DNA triangles with distinct DNA duplexes as edges and [Fe(tpy)2] vertices [Fig. 18(fc)] (169). Hybridization by slow cooling of 1 1 mixtures of two-arm DNA junctions based on bpy-Ru " or tpy-Ru complexes that had intramolecularly identical but intermolecularly complementary DNA arms led to infinite, bnear DNA polymer formation (170, 172). In contrast, room temperature hybridization of the same two-arm DNA junctions based on bpy-Ru + led to the formation of a mixture of structures, the majority of which were dimeric and cyclic [Fig. 18(c)] (172). 

Fig. 7. Use of FRET to show that the overall geometry of the four-way DNA junctions is a right-handed noncrossed structure. The 5 end of the DNA strands are labeled with filled circles. Noncrossed and crossed structures generate antiparallel or parallel alignment of DNA sequences, shown by the arrows at right. The six possible end-to-end distances were measured by labeling the appropriate 5 ends with fluorescein (donor) or tetramethylrho-damine (acceptor) and monitoring energy transfer. (From Murchie et al. )...

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