Double-strand breaks are repaired by error-free homologous recombination or by relatively error-prone pathways that directly join broken ends. Both types of repair have been extensively studied in Saccharomyces cerevisiae using enzymes HO or I-SceI, which create breaks with 4-nt 3′ overhangs. In the current study, a galactose-regulated zinc-finger nuclease (ZFN) designed to cleave the Drosophila rosy locus was used to generate breaks with 4-nt 5′ overhangs at out-of-frame cleavage sites inserted into the yeast LYS2 gene. Mutagenic repair was examined following selection of prototrophs on lysine-deficient medium containing galactose or surviving colonies on galactose-containing rich medium. Following cleavage of the original rosy spacer (ACGAAT), most Lys⁺ colonies contained 1- or 4-bp insertions at the cleavage site while most survivors had either a 2-bp insertion or a large deletion. Small insertions reflected nonhomologous end joining (NHEJ) and large deletions were the product of microhomology-mediated end joining (MMEJ). Changing the original ACGAAT spacer to either AGCAAT, ACGCGT or CTATTA altered the molecular features of NHEJ events as well as their frequency relative to MMEJ. Altering the optimal 6-bp spacer size between the zinc-finger protein binding sites to 5 bp or 7 bp eliminated the effect of continuous ZFN expression on survival, but Lys⁺ prototrophs were still generated. Analysis of Lys⁺ revertants after cleavage of the 5-bp spacer indicated that both the position and spacing of ZFN-generated nicks were variable. Results provide insight into effects of overhang sequence on mutagenic outcomes and demonstrate ZFN cleavage of 5- or 7-bp spacers in vivo.

双链断裂通过无错误同源重组或通过直接连接断裂末端的相对容易出错的途径修复。两种类型的修复都已在酿酒酵母中使用酶 HO 或 I- Sce I 进行了广泛研究，它们会产生带有 4-nt 3' 悬垂的断裂。在当前的研究中，设计用于切割果蝇玫瑰色基因座的半乳糖调节锌指核酸酶 (ZFN) 用于在插入酵母LYS2基因的框外切割位点处产生带有 4-nt 5' 突出端的断裂。在含有半乳糖的缺乏赖氨酸的培养基上选择原养生物或在富含半乳糖的培养基上选择存活的菌落后，检查诱变修复。原始切割后rosy spacer (ACGAAT)，大多数 Lys ⁺菌落在切割位点包含 1 或 4-bp 插入，而大多数幸存者有 2-bp 插入或大缺失。小的插入反映了非同源末端连接 (NHEJ)，而大的缺失是微同源介导的末端连接 (MMEJ) 的产物。将原始 ACGAAT 间隔子更改为 AGCAAT、ACGCGT 或 CTATTA 改变了 NHEJ 事件的分子特征及其相对于 MMEJ 的频率。将锌指蛋白结合位点之间的最佳 6-bp 间隔区大小更改为 5 bp 或 7 bp 可消除连续 ZFN 表达对存活的影响，但仍会产生Lys ^{+原养细胞。}赖氨酸分析⁺5-bp 间隔子切割后的回复突变表明 ZFN 生成的切口的位置和间距都是可变的。结果提供了对悬垂序列对诱变结果的影响的深入了解，并证明了 ZFN在体内对 5-或 7-bp 间隔区的切割。