Novel insights into ATP-Stimulated Cleavage of branched DNA and RNA Substrates through Structure-Guided Studies of the Holliday Junction Resolvase RuvX

•MtRuvX is a dimer in the asymmetric unit and each monomer has an RNase H fold.•MtRuvX has an intrinsic ATPase activity which is stimulated by DNA.•MtRuvX displays higher affinity to branched DNA species and RNA.•The Holliday junction cleavage activity is tightly coupled to ATP hydrolysis.•ATP hydrolysis switches MtRuvX to a DNA/RNA non-sequence specific endonuclease.