Figure 6.

High NaCl concentrations can partially suppress the recombination defective phenoytpe of the mutations. Panel (A) shows the appearance of products from recombination assays using T-15C:C+15G, G-16T:G+16A, G-16T and T-15C as substrates after incubation in either 500 mM or 1 M NaCl. Plasmids encoding the wild-type attB (wt), or the above mutants were incubated with pRT702 (attP) in recombination buffer adjusted to 100 mM, 500 mM or 1 M NaCl. After digesting with HindIII the DNA was separated in an agarose gel. The appearance of the 5435 bp fragment encoding attL is indicative of recombination. Panel (B) shows the synapse assays using the wild-type attB (wt), C-2A:G+2T, T-15C:C+15G and G-16T:G+16A under different NaCl conditions with wild type (left panel) or S12A integrase (right panel) with labelled attP. The complexes are annotated as described in Figure 4. −/+15 and −/+16 mutant attB sites accumulated both the cleaved complex (Int:cleaved attP/B), the shifted products (Int:attL/R) and released some free product (attL/R) with 500 mM and 1 M NaCl with the wild-type integrase. The synapse however as indicated using the S12A integrase did not become more abundant in high NaCl buffer, if anything it reduced. Panel (C) shows that the binding affinity of attB sites for integrase in the presence of different NaCl concentrations. Radiolabelled attB sites were incubated in binding buffer containing 50 mM, 500 mM or 1 M NaCl. Integrase was added at 66 nM. The shifted attB complexes are indicated by arrows. Only at 1 M NaCl, there was a slight increase in the free DNA for all four attB sites.