Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).
Sequence-specific double-strand cleavage of DNA by penta-N-methylpyrrolecarboxamide-EDTA X Fe(II).
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五-N-甲基吡咯甲酰胺-EDTA X Fe(II) 对 DNA 进行序列特异性双链切割。
DOI：
10.1073/pnas.80.22.6834
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发表时间：
1983
影响因子：
11.1
通讯作者：
Dervan,PB
中科院分区：
文献类型：
--
作者：
Schultz,PG;Dervan,PB
在O2和5 mM二硫苏糖醇存在下，0.5 μ M的五-N-甲基吡咯甲酰胺-EDTA X Fe（II）[P5 E X Fe（II）]在相反链上切割pBR 322质粒DNA（50 μ M碱基对），得到离散的DNA片段，如通过琼脂糖凝胶电泳分析的。对含有一个主要切割位点的~（32）P末端标记的517个碱基对限制性片段进行高分辨率变性凝胶电泳，结果表明，P_5E X Fe（II）切割3-5个碱基对，与6个碱基对的序列5 ′-T-T-T-T-T-A-3 ′（4，323 - 4，328个碱基对）相邻。五肽的主要结合方向发生在该序列腺嘌呤侧的氨基末端。在5 mM二硫苏糖醇存在下，0.01 μ M P5 E X Fe（II）将0.22 μ M质粒（1.0 mM碱基对）的I型pBR 322 DNA转化为40%的II型，表明切割反应是催化性的，最少转化9次。该合成分子在6个碱基对识别水平上实现DNA的双链切割（pH 7.9，25 ℃），并可能提供一种设计“人工限制性内切酶”的方法。
In the presence of O2 and 5 mM dithiothreitol, penta-N-methylpyrrolecarboxamide-EDTA X Fe(II) [P5E X Fe(II)] at 0.5 microM cleaves pBR322 plasmid DNA (50 microM in base pairs) on opposite strands to afford discrete DNA fragments as analyzed by agarose gel electrophoresis. High-resolution denaturing gel electrophoresis of a 32P-end-labeled 517-base-pair restriction fragment containing a major cleavage site reveals that P5E X Fe(II) cleaves 3-5 base pairs contiguous to a 6-base-pair sequence, 5'-T-T-T-T-T-A-3' (4,323-4,328 base pairs). The major binding orientation of the pentapeptide occurs with the amino terminus at the adenine side of this sequence. In the presence of 5 mM dithiothreitol, 0.01 microM P5E X Fe(II) converts form I pBR322 DNA at 0.22 microM plasmid (1.0 mM in base pairs) to 40% form II, indicating the cleavage reaction is catalytic, turning over a minimum of nine times. This synthetic molecule achieves double-strand cleavage of DNA (pH 7.9, 25 degrees C) at the 6-base-pair recognition level and may provide an approach to the design of "artificial restriction enzymes".