Figure 5

A118 integrase binding to different att sites. Gel mobility shift assays with increasing amounts of integrase and ³²P-labeled 100 bp fragments containing (A) attP, (B) attB, (C) attL, and (D) attR, respectively. Integrase concentrations were 0, 5, 10, 30, 90, 270, and 810 nM. Assignments of monomer and dimer DNA complexes were based on their migrations relative to complexes formed by the integrase C-terminal domain (for example, (E),(F)) and in CHAPS titration experiments (for example, (G)). Apparent dissociation constants (K_d values) for the dimer complexes (mean and standard deviation from three experiments) are given below each panel. (E) Gel mobility shift assay employing integrase subjected to partial proteolysis with chymotrypsin (0, 20, 40, and 60 ng for 10 minutes) and ³²P-labeled attP fragments (lanes 2 to 5). The chymotrypsin-digested integrase samples used for this experiment appeared nearly identical by SDS gel electrophoresis to those in Figure 2C, lanes 1 to 3. Lanes 1 and 6, controls without protein added; lane 7, reaction with the purified recombinant C-terminal integrase domain (30 nM). (F) Gel mobility shift assays with increasing amounts of the recombinant integrase C-terminal domain and ³²P-labeled 100 bp fragments containing attP or attB. Integrase C-terminal domain concentrations were 0, 5, 10, 30, 90, 270, and 810 nM. (G) Effect of CHAPS on integrase binding to att sites. Integrase (90 nM) was incubated with attB and attP in the presence of 0, 5, 10, 15, and 20 mM CHAPS. No integrase was added to the left lanes of each panel. (H) Effect of CHAPS on integrase recombination activity. Intramolecular attP×attB deletion reactions (15 minutes) were performed in the presence of 0, 2, 4, 6, 8, and 10 mM CHAPS.