FIG. 4.

Analysis of nick-defective and hairpin-defective mutants under relaxed RSS cleavage conditions in Mn²⁺. The 10 mutants showing defective nicking and/or hairpin formation in the coupled cleavage assay were tested for cleavage of a radiolabeled 12-RSS substrate under relaxed cleavage conditions (in Mn²⁺). Mutants are grouped and labeled according to their phenotypes in Mg²⁺. Three mutants (R621, R713, and H795) remained defective for nicking (lanes 2 to 4), and three mutants remained specifically defective for hairpin formation (lanes 8 to 10). Interestingly, these hairpin-defective mutants made low levels of aberrant hairpins, indicated by arrows. Representative data are shown. All lanes are from the same exposure of the same gel but have been rearranged for clarity. The positions of the uncleaved substrate, the nick product, and the normal hairpin product are indicated by diagrams on the left. The star indicates the position of the radiolabel. WT, wild type.