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首页> 外文期刊>Molecular and Cellular Biology >Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases
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Stimulation of Homologous Recombination through Targeted Cleavage by Chimeric Nucleases

机译：嵌合核酸酶通过靶向切割刺激同源重组。

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Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.
机译：测试了在非特异性DNA切割域和锌指DNA识别域之间杂交的嵌合核酸酶在活细胞中发现和裂解靶位点的能力。将工程化的DNA底物和核酸酶都注射到非洲爪蟾卵母细胞核中，观察到DNA裂解和随后的同源重组。特异性切割需要锌指识别位点的两个反向拷贝紧密相邻，这反映了对切割域的二聚化的需要。切割的DNA分子被激活进行同源重组;在最佳条件下，即使将DNA组装成染色质，基本上100％的底物也会重组。最初的核酸酶在锌指和切割结构域之间具有一个18个氨基酸的连接子，并且该酶在卵母细胞中以6至18 bp的间隔区隔开的成对位点切割，最理想的是8 bp。通过缩短接头，我们发现有效位点分离的范围可以大大缩小。在结合结构域和切割结构域之间没有故意的接头的情况下，只有恰好相距6bp的结合位点支持卵母细胞中的有效切割。我们还显示，当适当地放置两个嵌合酶时，具有不同结合特异性的两种嵌合酶可以协同刺激重组。由于锌指的识别特异性可以通过实验改变，因此这种方法在诱导多种生物的靶向重组方面具有广阔的前景。

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