Mutagenic repair of a ZFN-induced double-strand break in yeast: Effects of cleavage site sequence and spacer size.
Mutagenic repair of a ZFN-induced double-strand break in yeast: Effects of cleavage site sequence and spacer size.
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DOI：
10.1016/j.dnarep.2021.103228
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发表时间：
2021-12
期刊：
影响因子：
3.8
通讯作者：
Jinks-Robertson S
中科院分区：
文献类型：
--
作者：
Shaltz S;Jinks-Robertson S
双链断裂可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。这两种类型的修复已经在酿酒酵母中使用酶HO或I-SceI进行了广泛的研究，它们产生具有4-nt 3 '悬垂的断裂。在目前的研究中，设计了一种半乳糖调节的锌指核酸酶（ZFN），用于切割果蝇玫瑰色位点，在插入酵母LYS2基因的框外切割位点产生具有4-nt 5 '悬垂的断裂。在含有半乳糖的赖氨酸缺乏培养基或含有半乳糖的富培养基上选择存活菌落后，研究了原营养体的诱变修复。在原始玫瑰色间隔区（ACGAAT）切割后，大多数Lys+菌落在切割位点含有1-或4-bp的插入，而大多数存活的菌落要么有2-bp的插入，要么有较大的缺失。小插入反映了非同源末端连接（NHEJ），大缺失是微同源介导的末端连接（MMEJ）的产物。将原来的ACGAAT间隔改为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间隔长度6 bp调整为5 bp或7 bp，消除了连续表达ZFN对存活的影响，但仍会产生Lys+原生营养物。裂解5bp间隔后的Lys+回复性分析表明，zfn产生的切口的位置和间距都是可变的。研究结果揭示了悬垂序列对诱变结果的影响，并证明了ZFN在体内可切割5-或7-bp间隔物。
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.
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