Figure 20.

Beta-promoted Recombineering with ssDNA oligos. (A–C) Targeting of the lagging strand template. Beta promotes annealing of the oligo to a ssDNA region of the lagging strand template. Pol I and ligase promote filling in of the oligo and joining it with the surrounding Okazaki fragments to produce heteroduplex DNA (red asterisk). (D–F) Targeting of the leading strand template. Beta promotes annealing of the oligo to a ssDNA region of the leading strand template just ahead of the leading strand 3′ end. The leading strand polymerase (not shown) dissociates from its template, and because it is tethered to the clamp loader (green pentagon) of the replisome (denoted by yellow oval), it can reinitiate downstream at the 3′ end of the oligo. Annealing of the oligo creates either a mismatch, a small deletion, or small insertion (denoted by red asterisk), which must escape repair by the Mismatch Repair System of E. coli. The mutation is fixed via subsequent replication of the heteroduplex region.