(A) Overall view of the XerH-difHLP structure in cartoon representation. Sequence of the difHLP substrate is shown above (written in the 3’ to 5’ direction, with the arms in gold, central region in gray) the nick positions are marked by triangles. Figure 4—figure supplement 1 shows a snapshot of the electron density map. (B) Compared to the pre-cleavage XerH-difH complex, both subunits (A, gold; B, blue surface) have rotated ~22° towards each other, concomitant with DNA bending. Insert: Close-up of DNA bending: A17’ and G18’ (red) are unstacked, with a 90° kink. (C) XerH (left) interacts with the DNA kink differently than Cre (PDB: 1NZB; right). The electrostatic surface potential is shown in red (negative) and blue (positive). (D) Superimposition of the two subunits (A, gold and B, blue) bound to the same difHLP site illustrates their different conformations. Left insert: Helices αN and αO are repositioned relative to the pre-cleavage structure (grey), including rotations (marked by arrows) and shortening of the helices (red dashed lines showing the distances between Cα of L338 and Cα of W359). Right insert: repositioning of the β2-β3 loop upon activation enables the catalytic K239 to interact with the DNA. For clarity, only the DNA associated with the golden monomer is shown. (E) Active site conformations of the distinct XerH subunits. In the active subunit (molecule A, left), R213, H309, R312, and H335 make hydrogen bonds with the scissile phosphate (orange), K239 contacts the base of the adjacent nucleotide A12, and Y344 is covalently attached to the DNA. In the inactive subunit (right) the catalytic tyrosine is 5.5 Å away from the scissile phosphate, R213 and H335 point away, and the K239 side chain is disordered. Red sphere – bound water molecule; dashed lines – hydrogen bonds.