Crystal Structure of a Eukaryotic GEN1 Resolving Enzyme Bound to DNA

We present the crystal structure of the junction-resolving enzyme GEN1 bound to DNA at 2.5 Å resolution. The structure of the GEN1 protein reveals it to have an elaborated FEN-XPG family fold that is modified for its role in four-way junction resolution. The functional unit in the crystal is a monomer of active GEN1 bound to the product of resolution cleavage, with an extensive DNA binding interface for both helical arms. Within the crystal lattice, a GEN1 dimer interface juxtaposes two products, whereby they can be reconnected into a four-way junction, the structure of which agrees with that determined in solution. The reconnection requires some opening of the DNA structure at the center, in agreement with permanganate probing and 2-aminopurine fluorescence. The structure shows that a relaxation of the DNA structure accompanies cleavage, suggesting how second-strand cleavage is accelerated to ensure productive resolution of the junction. [Display omitted] •GEN1 crystallized with a resolution product containing two perpendicular DNA helices•GEN1 shares the FEN1 superfamily fold, with a two-metal ion-containing active site•GEN1 forms a dimer that juxtaposes two products in a substrate-like complex•A resulting model of a GEN1-junction complex is supported by solution experiments Liu et al. present the crystal structure of a fungal GEN1 Holliday junction-resolving enzyme. GEN1 is bound to a product of cleavage, comprising two connected arms of the junction. Two GEN1 molecules dimerize to juxtapose two products such that they can be simply reconnected to form a junction.