Figure 3.

Rate of recombination by wt versus E449K integrases. (A) A time course of attP x attB recombination and direct detection of recombinants by restriction and agarose gel electrophoresis. The time course was performed at 15°C to slow recombination with 733 nM integrase and reactions stopped by incubation at 80°C, 10 min. Reactions were digested with HindIII. The product of integration, pRT602700, gave two fragments, 5435 bp (attL) and 91 bp (attR; data not shown), resulting from the fusion of the substrates, 3034 bp (pRT700, attP) and 2513 bp (pRT602, attB). (B) Time course of attP x attB recombination using linear DNA fragments. Purified integrases (733 nM) were incubated with radio-labelled attP (98 bp; 1.5 nM) and a 235 bp fragment encoding attB (13 nM) for a time series at 30°C. The reactions were stopped by heating (80°C, 10 min) and then digested with subtilisin before loading onto a 5% polyacrylamide gel. The gel was dried and the radioactivity assayed using a phosphorimager. The positions of free probe (attP) the products of recombination (attL and attR) and the cleaved attP fragments (attP′ 57 bp and attP′ 41 bp) are indicated. (C) The amount of radioactivity in the products (black lines) versus the cleaved substrates (grey lines) was quantified and plotted against time for (i) attB x attP with wt integrase, (ii) attB x attP E449K integrase.