Figure 2. WHO6 induces allele-specific DNA cleavage of the T. delbrueckii FBA1 gene, with DNA repair by gene conversion or NHEJ.

(A) Summary of the experiment. Haploid S. cerevisiae strains, containing a non-expressed T. delbrueckii FBA1 ORF (TdFBA1-S or TdFBA1-R alleles) integrated at ADE2, were transformed multiple independent times with plasmids expressing WHO6 or WHO6-HA. In all transformations of strains containing the TdFBA1-S allele, the only colonies that survived expression of WHO6 were ones in which TdFBA1-S underwent DNA cleavage and repair by gene conversion or imprecise NHEJ, changing its sequence and making it resistant to further cleavage. (B) Gene conversion and imprecise NHEJ events in TdFBA1-S. The reference DNA sequence (uppercase) shows the 3’ end of the TdFBA1-S allele from T. delbrueckii strain CBS1146. Survivors 1–5 are transformants in which TdFBA1-S was cleaved by Who6 and repaired by imprecise NHEJ near position 668 (green box and triangle; survivors 1 and 2 have a 1 bp insertion, and survivors 3–5 have a 1 bp deletion, relative to the sequence TTT in the reference). Survivors 6–12 are transformants in which TdFBA1-S was cleaved by Who6 and partially overwritten by gene conversion with the endogenous S. cerevisiae FBA1 gene. Gene conversion regions are highlighted with pink backgrounds. The TdFBA1-R allele from T. delbrueckii strain L09, which is the natural host of WHO6, is also shown; this allele acquired no sequence changes among 10 independent pWHO6 transformants examined. A putative Who6 recognition site (yellow) and cleavage site with 4 bp 3’ overhang (underlined) are marked. Survivors 10 and 11 are from transformations with pWHO6-HA; all other survivors are from transformations with pWHO6. The complete TdFBA1 gene was sequenced from all transformants but only positions 616 to 975 are shown; there were no changes outside this region.