Difference topology: The DNA path within a high-order DNA-protein assembly can be revealed from the topology of DNA knots and links formed by the action of Flp or Cre site-specific recombinase on target sites placed close to (and flanking) the assembly. This ‘analysis is experimentally simple, and has broad applicability. Choice between alternative reaction mechanisms: Recombination product topologies resulting from a fixed synaptic topology can help distinguish between alternative modes of site arrangement and strand exchange by a given recombinase enzyme. Conserved biological functions revealed by conserved topology of DNA loci in vivo: DNA topology can reveal evolutionary relationships between two diverged DNA loci that perform analogous but distinct biological functions. The unusual positive chromatin writhe at the centromeres of yeast chromosomes is shared by the partitioning locus of an extrachromosomal circular DNA element present in the yeast nucleus. This finding lends credence to the possible origin of these loci from a common ancestor that once directed the segregation of both the chromosomes and the plasmid during cell division. Chemical chirality in DNA recombination: The active site of the Flp/Cre recombinase contains two conserved positively charged side-chains (Arg-I and Arg-II) responsible for compensating the negative charge on the non-brridging oxygen atoms of the scissile phosphate group. When one of the oxygens is replaced by a neutral group, one of the arginine residues becomes dispensable. The reactivity of DNA substrates containing substitutions at either of the two oxygens with Flp/Cre lacking either of the two arginines reveals the chirality of Arg-oxygen interactions, and defines the stereochemical course of the recombination reaction.