Figure 3.

Activities and crosslinking of helix D mutants. (A) Inversion of DNA between the hix sites on pMS551 by Hin reorients the HindIII site to produce different sized fragments on an agarose gel when co-digested with Pst I (32). Time courses were performed with the indicated Hin mutant together with Fis and HU on supercoiled pMS551, and aliquots were taken at the times indicated. Hin–H107Y, H107Y/K72C and H107Y/A76C exhibit similar inversion kinetics. (B) Synaptic complex assembly on oligonucleotide substrates. Hin was incubated with 36 bp 3′ ³²P-labeled DNA for 20 min and electrophoresed on a native polyacrylamide gel containing 10% glycerol to enhance synaptic complex formation. Each of the mutants forms large amounts of synaptic complexes. (C) Crosslinking of mutants with cysteines in helix D. Reactions were performed as in panel B and then subjected to crosslinking for 1 min. Synaptic complexes were excised from a native gel, eluted and crosslinked products were separated by SDS–PAGE. Crosslinkers were diamide (0 Å), BMOE (8 Å spacer) and BMH (16 Å spacer). The locations of non-crosslinked and crosslinked Hin–DNA(³²P) covalent complexes are shown on the autoradiograph.