In vivo evidence for the ssDNA intermediate. (A) Schematic representations of recombination reactions to replace the ampicillin (bla) with the blasticidin (bsd) resistance gene in either the E. coli chromosome or pBelo-Bac11. The bla gene was present in both orientations (bla, inv-bla) to alter its orientation to the direction of replication, which is indicated by an arrow beside the origin, either oriC or oriS. The bsd gene was PCR amplified to possess different 5' ends as indicated, either phosphorylated (P, black stars), hydroxylated (O, no symbol) or phosphothioated (S, open circles) in various combinations. PSSP indicates that both 5' ends were phosphorylated and the first two backbone linkages were also phosphothioated. All three Red proteins (α, β, γ) were expressed in these experiments. (B) SSCP-PAGE gel of a hydroxylated/phosphorylated linear dsDNA substrate (OP) after transformation in vivo. The lower band represents dsDNA (ds) and the upper bands represent the slower migrating secondary structures of the single-strands after heat denaturation (up and low). DNA was harvested from cells immediately after electroporation (OP) or after incubation for 5 minutes at 37°C (OP5'). (C, D) Quantification of recombinant colonies using the experimental design shown in (A). The targeted loci were either located on the E. coli chromosome (C) or on the pBelo-Bac11 BAC (D) in direct (bla) or inverted (inv-bla) orientation. (E) Kanamycin-resistant colonies after repair of pBelo-Bac11-neo* BAC using dsDNA with zero (OP), one (SP-1), two (SP-2), four (SP-4) or six (SP-6) consecutive phosphothioated bonds at one 5' end. The other 5' end was phosphorylated (P).