Junction resolving enzymes use multivalency to keep the Holliday junction dynamic

Holliday junction (HJ) resolution by its resolving enzymes is essential for chromosome segregation and recombination-mediated DNA repair. HJs undergo two types of structural dynamics that determine the outcome of recombination: conformer exchange between two isoforms and branch migration. However, it is unknown how the preferred branch-point and conformer are achieved between enzyme binding and HJ resolution given the extensive binding interactions seen in static crystal structures. Single molecule fluorescence resonance energy transfer analysis of resolving-enzymes from bacteriophages (T7 endonuclease I), bacteria (RuvC), fungi (GEN1) and humans (hMus81-Eme1) showed that both types of HJ dynamics still occur after enzyme binding. These dimeric enzymes use their multivalent interactions to achieve this, going through a partially-dissociated intermediate in which the HJ undergoes nearly unencumbered dynamics. This evolutionarily conserved property of HJ resolving-enzymes provides previously unappreciated insight on how junction resolution, conformer exchange and branch migration may be coordinated.