FIG. 5.

Prenicked oligonucleotide substrates specifically assay for the hairpin formation step. Mutants that exhibited a nicking defect (lanes 2 to 4) or aberrant hairpin formation (lanes 5 to 7) under relaxed cleavage conditions were tested for hairpin formation with a prenicked substrate in Mn²⁺, a strategy which bypasses the requirement for the generation of a nick at the correct position. The position of the aberrant hairpin is indicated by an arrow. Representative data are shown. All lanes are from the same exposure of the same gel but have been rearranged for clarity. Lane 5 is overloaded, but quantification reveals ratios of products and substrate similar to those in other lanes. See the legend to Fig. 3 for explanations of symbols. WT, wild type.