Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

Abstract

DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains.

Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

“…Our search for HC-specific binding proteins required to have available radiolabeled DNA HCs. These DNA structures were built as described in [20]. The starting material was two distinct radiolabeled dsMCs, one of 215 base pairs (dsMC10) and one of 235 base pairs (dsMC09), produced after circularization of 32 P-end labeled fragments ( Fig EV1).…”

“…HCs were prepared as described in [20] with three modifications in the procedure. Firstly, the production of dsMCs was performed in the presence of ethydium bromide (0.35 µg/mL) to favor circularization of DNA fragments into monomeric circles.…”

“…Our goal in this work was to explore further these hypotheses by seeking nuclear proteins capable of stabilizing HC structures by specifically binding to them. We took advantage of the existence of a procedure to build HCs from two distinct double stranded DNA mini-circles (dsMCs) [20] to fractionate nuclear extracts from Hela cells over several chromatographic media. This approach made possible the identification of three RNA-binding proteins capable of binding specifically to HC: the Tudor-Staphylococcal Nuclease Domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing (DBHS) family, the ParaSpeckle Protein Component 1 (PSPC1) and the Splicing Factor Proline-and Glutamine-rich protein (SFPQ).…”

“…The single stranded catenanes were gel-purified and re-annealed with the C09ss and C10ss using the wheat germ Topoisomerase I to finally get the DNA HCs. Adapted from [20].…”

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures

“…[1][2][3][4] Among the many nanostructures fabricated from DNA, single-stranded DNA rings are characterized by dynamic and mobile features which cannot be otherwise fullled, and they are one of the most important and promising parts in nanoarchitectonics. [5][6][7] For example, catenanes, in which two or more DNA rings are mechanically interlocked, have been elegantly employed in molecular switches, molecular motors, and switchable catalysts. [7][8][9][10] Versatile applications of DNA rings to molecular biology, medicine, and biotechnology have also been reported.…”