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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Structure-guided reprogramming of serine recombinase DNA sequence specificity
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Structure-guided reprogramming of serine recombinase DNA sequence specificity

机译：结构指导的丝氨酸重组酶DNA序列特异性重编程

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摘要

Routine manipulation of cellular genomes is contingent upon the development of proteins and enzymes with programmable DNA sequence specificity. Here we describe the structure-guided reprogramming of the DNA sequence specificity of the invertase Gin from bacteriophage Mu and Tn3 resolvase from Escherichia coli. Structure-guided and comparative sequence analyses were used to predict a network of amino acid residues that mediate resolvase and invertase DNA sequence specificity. Using saturation mutagenesis and iterative rounds of positive antibiotic selection, we identified extensively redesigned and highly convergent resolvase and invertase populations in the context of engineered zinc-finger recombinase (ZFR) fusion proteins. Reprogrammed variants selectively catalyzed recombination of nonnative DNA sequences >10,000-fold more effectively than their parental enzymes. Alanine-scanning mutagenesis revealed the molecular basis of resolvase and invertase DNA sequence specificity. When used as rationally designed ZFR heterodimers, the reprogrammed enzyme variants site-specifically modified unnatural and asymmetric DNA sequences. Early studies on the directed evolution of serine recombinase DNA sequence specificity produced enzymes with relaxed substrate specificity as a result of randomly incorporated mutations. In the current study, we focused our mutagenesis exclusively on DNA determinants, leading to redesigned enzymes that remained highly specific and directed transgene integration into the human genome with >80% accuracy. These results demonstrate that unique resolvase and invertase derivatives can be developed to site-specifically modify the human genome in the context of zinc-finger recombinase fusion proteins.
机译：细胞基因组的常规操作取决于具有可编程DNA序列特异性的蛋白质和酶的开发。在这里，我们描述了来自噬菌体Mu的转化酶Gin和来自大肠杆菌的Tn3分解酶的DNA序列特异性的DNA结构指导。使用结构指导和比较序列分析来预测介导分辨酶和转化酶DNA序列特异性的氨基酸残基网络。使用饱和诱变和阳性抗生素选择的迭代回合，我们在工程化锌指重组酶（ZFR）融合蛋白的背景下，确定了经过重新设计且高度收敛的分辨酶和转化酶种群。重新编程的变体比其亲本酶更有效地催化> 10,000倍的非天然DNA序列重组。丙氨酸扫描诱变揭示了分辨酶和转化酶DNA序列特异性的分子基础。当用作合理设计的ZFR异二聚体时，重新编程的酶变体会进行位点特异性修饰的非天然和不对称DNA序列。早期对丝氨酸重组酶DNA序列特异性进化的研究，由于随机掺入突变，产生了具有松弛底物特异性的酶。在当前的研究中，我们仅将诱变集中在DNA决定簇上，从而导致重新设计的酶保持高度特异性，并以高于80％的准确性指导转基因整合到人类基因组中。这些结果表明，在锌指重组酶融合蛋白的背景下，可以开发独特的分辨酶和转化酶衍生物来定点修饰人类基因组。

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    Thomas Gaj; Andrew C. Mercer; Charles A. Gersbach; Russell M. Gordley; Carlos F. Barbas Ill;

  • 作者单位

    The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

    The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

    The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

    The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

    The Skaggs Institute for Chemical Biology and the Departments of Molecular Biology and Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    gene targeting; protein engineering; site-specific recombination; zinc-finger recombinase;

    机译：基因靶向蛋白质工程;位点特异性重组;锌指重组酶;

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