Stoichiometric incorporation of base substitutions at specific sites in supercoiled DNA and supercoiled recombination intermediates

Supercoiled DNA is the relevant substrate for a large number of DNA transactions and has additionally been found to be a favorable form for delivering DNA and protein-DNA complexes to cells. We report here a facile method for stoichiometrically incorporating several different modifications at multiple, specific, and widely spaced sites in supercoiled DNA. The method is based upon generating an appropriately gapped circular DNA, starting from single-strand circular DNA from two phagemids with oppositely oriented origins of replication. The gapped circular DNA is annealed with labeled and unlabeled synthetic oligonucleotides to make a multiply nicked circle, which is covalently sealed and supercoiled. The method is efficient, robust and can be readily scaled up to produce large quantities of labeled supercoiled DNA for biochemical and structural studies. We have applied this method to generate dye-labeled supercoiled DNA with heteroduplex bubbles for a Förster resonance energy transfer (FRET) analysis of supercoiled Holliday junction intermediates in the λ integrative recombination reaction. We found that a higher-order structure revealed by FRET in the supercoiled Holliday junction intermediate is preserved in the linear recombination product. We suggest that in addition to studies on recombination complexes, these methods will be generally useful in other reactions and systems involving supercoiled DNA.