Figure 1.

ZFN cleavage of spacer ACGAAT and error-prone repair events. (A) The sequence of the 24 bp rosy sequence inserted into LYS2 is shown, with the three bases recognized by each zinc finger (blue or red ovals for RyA and RyB, respectively) indicated. The yellow ovals represent the dimerized FokI domains; the 6-bp spacer is in red font and triangles indicate positions of enzyme-generated nicks that create 4-bp 5’ overhangs. (B) Sequences added during repair events are in lowercase red. Only the first three classes (+CGAA, 2A>3A and 1T>2T) generate Lys⁺ revertants; the fourth class (+AC) was only observed only among survivors. Complete filling of ends duplicates the region bounded by the ZFN nicks (+CGAA) while out-of-register pairing between an A and T in the overhangs generates the 2A>3A mutation. Duplication of the T (1T>2T) adjacent to the distal overhang can be generated by misincorporation-realignment, followed by ligation of the re-created 4-nt overhangs. The +AC event can be generated by a similar misincorporation-realignment mechanism during initial filling of the proximal overhang. (C) Mechanism for the NHEJ-dependent 29-bp deletion that is frequent among Lys⁺ revertants in the pol4Δ background. Resection of the 5’ ends allows pairing between GCC repeats (red) that flank the DSB. Subsequent removal of 3’ tails and filling of flanking gaps (red lowercase) completes repair.