Figure 3

Enzymatic activities of WT and mutant INs. (A) 3' processing and strand transfer activities of IN mutants. Using an oligonucleotide-based assay, activities of recombinant INs were tested at two different concentrations: 100 nM (lanes 2, 4, 8, 10, 12); or 120 nM (lanes 3, 5, 7, 9, 11, 13). Reaction products were separated in a denaturing urea gel and visualized with a STORM PhosphorImager (Molecular Dynamics). (B) Quantification of 3'-processing and strand transfer activities of IN mutants. Gels from figure 3A were quantified with Image Quant™ 4.1 software. (C) Concerted integration assay was performed using IN (2 pmoles, lanes 1, 3, 5, 7, 9, 11 and 5 pmoles, lanes 2, 4, 6, 8, 10, 12), 150 ng of naked acceptor DNA (3000 bp) and 15 ng of ³²P 5'-labelled donor pre-processed DNA (296 bp). The reaction products were loaded on 1% agarose. The positions of the different products obtained after half-site integration (HSI), full-site integration (FSI) and donor/donor integration (d/d) are reported.