Abstract
Structure-selective nucleases perform DNA strand incisions crucial to the repair/resolution of branched DNA molecules arising during DNA replication, recombination, and repair. From a combination of genetics and in vitro nuclease assay studies, we are just beginning to understand how these enzymes recognize their substrates and to identify their in vivo DNA structure targets. By performing nuclease assays on a variety of substrates meant to mimic cellular intermediates, structural features of branched DNA molecules that are important for robust catalysis can be defined. However, since these enzymes often are capable of cleaving a range of DNA structures, caution must be taken not to overemphasize the significance of incision of a certain structure before a careful and detailed kinetic analysis of a variety of DNA substrates with different polarities and structural features has been completed. Here, we provide protocols for the production of a variety of oligo-based DNA joint molecules and their use in endonuclease assays, which can be used to derive the kinetic parameters KM and kcat. Determination of these values for a variety of substrates provides meaningful comparisons that allow inferences to be made regarding in vivo DNA structure target(s).