Figure 1. Redβ requires both N- and C-terminae for homologous recombination.

(a) Diagram of Redβ depicting the CR1–3 regions conserved with other phage SSAPs⁵ as well as the deletions and point mutations used in this work. (b) Expression of Redβ from pSC101BAD-Redβ induced by arabinose and evaluated by Western using a Redβ antibody. Lane Wt Redβ-; uninduced with arabinose: lane pSC101 empty; induced with arabinose but without Redβ in the expression plasmid. (c) Schematic representation of the ssOR (single strand Oligonucleotide Repair) assay. The neomycin resistance gene was mutated to introduce a central stop codon (neo*) and inserted into a BAC. Repair of the mutation by incorporation of an oligonucleotide restores kanamycin resistance. The complementary single strand oligonucleotides can either anneal to the lagging strand template and act as an Okazaki-like fragment primer (LG) or not (LD). The BAC resides in an E. coli host, which is induced for expression of wt and mutant Red proteins from a pSC101 plasmid by addition of arabinose 45 minutes before electroporation of the oligonucleotide followed by quantitating the acquisition of kanamycin resistance by counting colonies on kanamycin plates. (d) Performance of wt and truncated Redβ proteins in the ssOR assay using either the LD oligo (light blue) or LG oligo (dark blue). Experiments were performed in triplicates and data were normalized to the number of cells transformed with a control plasmid and are represented as mean ± SD. (e) Schematic representation of the Beta recombination assay. Two PCR products were generated to carry the neo (kanamycin resistance) gene. Both products carried identical DNA sequence however differed according to the position of two consecutive phosphothioate bonds at opposing 5′-ends. In both PCR products, the other 5′ end was phosphorylated. After electroporation, only one strand will be digested by Redα into full length single strands as illustrated. The strand whose 3′ end can serve as a primer for Okazaki-like fragment synthesis is termed ssLG and the other strand is termed ssLD. The kanamycin resistance gene (kan) is flanked by 50 bp homology arms (purple and green) that flank the ampicillin resistance gene in pBAD. (f) Performance of the wt and truncated Redβ proteins in the Beta recombination assay. Experiments were performed as described in (d).