RecD is the general Holliday junction resolvase in Gram-positive bacteria and is 2 required for efficient homologous DNA recombination and repair. X-ray crystallographic techniques and biochemical experiments have been employed to give insight into RecD-Holliday junction binding and its interaction with other known proteins in this DNA repair pathway. The 2.2 A crystal structure of Bacillus subtilis RecU was determined using multiple isomorphous replacement. In addition, structures containing divalent cation and the D88A variant were also determined. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. Surface plasmon resonance and biochemical assays were used to investigate the biochemical properties of RecD, RuvA and the proposed RecUlRuvNHolliday junction complex. The RecD structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. The structure suggests a model for the way in which a Holliday junction may be bound that is consistent with biochemical data. Surface Plasmon resonance experiments have highlighted the differences between the Escherichia coli and Bacillus subtilis RuvA proteins and the ability of RecU to bind with RuvA to form a RecUlRuvNHolliday junction complex.