ACTIVATION OF RUVC HOLLIDAY JUNCTION RESOLVASE IN-VITRO

The Escherichia coil RuvC protein is an endonuclease that resolves Hol liday junctions. In vitro, the protein shows efficient structure-speci fic binding of Holliday junctions, yet the rate of junction resolution is remarkably low. We have mapped the sites of cleavage on a syntheti c junction through which a crossover can branch migrate through 26 bp and find that greater than or equal to 90% of the junctions were cleav ed at one site. This observation of sequence-specific cleavage suggest s that inefficient resolution may be due to DNA binding events which o ccur away from the cleavage site and are therefore non-productive. Hol liday junction resolution by RuvC protein can be stimulated by a numbe r of factors including: (i) the presence of Mn2+ (rather than Mg2+) as the divalent metal cofactor, (ii) alkaline pH (less than or equal to 10), and (iii) elevated temperature. These observations may indicate t hat other proteins are required for efficient RuvC-mediated resolution .