Figure 5. Redβ C-terminal point mutations that decrease the Redα interaction impair Beta recombination but not ssOR.

(a) Schematic representation of the neo-tail assay. The stop codon of the neo gene was mutated to TGC. Homologous recombination to revert the stop codon to TGA will restore kanamycin resistance. Six dsDNA substrates were prepared, each including the same 35 bp homology arms (green and orange) and a pair of phosphothioate bonds at the 5′ end of the strand whose 3′ end can serve to prime Okazaki-like fragment synthesis. The six substrates differed by the lengths between the homology arms (10 + 70 = 80, 30 + 70 = 100, 50 + 70 = 120, 100 + 70 = 170, 150 + 70 = 220 and 200 + 70 = 270 bp) and were electroporated as either ds or ssDNA after in vitro Redα digestion. (b) Neo-tail assay comparison of the six substrates as ds (black) or ss (red) DNA in homologous recombination mediated by wild type Redβ. Redβ was either expressed from pSC101-BAD-Redβ (ss) or pSC101-BAD-Redγβα (ds) and the results are from three independent experiments each performed in triplicate. Data were normalized to the number of cells transformed with a control plasmid and are represented as mean ± SD. (c) Recombination efficiencies for Redβ Q252A, E256A and Redβ E191A, Q252A, E256A expressed as a ratio compared to wt Redβ using the neo-tail assay with ss and dsDNA substrates. Error bars indicate the mean ± SD of an experiment performed in triplicate.