The endonuclease Hje catalyses rapid, multiple turnover resolution of Holliday junctions.

Holliday junction-resolving enzymes are ubiquitous, structure-specific endonucleases that resolve four-way DNA junctions by the introduction of paired nicks in opposing strands, and are required for homologous recombination, double-strand break repair, recombination-dependent restart of stalled or collapsed DNA replication forks, and phage DNA processing. Here, we present the first steady-state kinetic characterisation of a junction-resolving enzyme; the Hje endonuclease from Sulfolobus solfataricus. We demonstrate that substrate turnover by Hje is sequence-independent and limited largely by the rate of cleavage of the phosphodiester bonds of the bound Holliday junction substrate, rather than substrate association or product dissociation. Reaction rates under multiple turnover conditions compare favourably with type II restriction enzymes. These properties, coupled with a high level of specificity for four-way junctions over all other DNA substrates, make Hje a suitable enzyme for applications requiring the detection and cleavage of Holliday junctions in vitro.