Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase.
Sequence-specific cleavage of DNA via nucleophilic attack of hydrogen peroxide, assisted by Flp recombinase.
复制标题
在 Flp 重组酶的协助下，通过过氧化氢的亲核攻击对 DNA 进行序列特异性切割。
DOI：
10.1021/bi00069a002
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发表时间：
1993
期刊：
影响因子：
2.9
通讯作者：
Tullius,TD
中科院分区：
文献类型：
--
作者：
Kimball,AS;Lee,J;Jayaram,M;Tullius,TD
当与来自酿酒酵母的重组酶Flp一起使用时，过氧化氢能够影响DNA中特定磷酸二酯键的断裂。这种切割不是由DNA骨架的氧化损伤引起的，而是过氧化物亲核攻击的结果。一个磷-氧键在反应中断裂。过氧化物对DNA的切割也发生在Flp的失活突变体中，其中活性位点亲核体酪氨酸被苯丙氨酸取代。除了提供信息链切割的Flp的机制，这些结果可能有助于开发新的合成DNA切割试剂，通过水解，而不是自由基chemical.The磷酸二酯骨架的DNA是众所周知的耐水解切割。因此，许多用于降解DNA的小分子通过自由基化学裂解脱氧核糖骨架（Stubbe & Kozarich，1987）。当然，酶能够有效地水解DNA的磷酸二酯键。我们在这里报告了一个系统，其中过氧化氢作为亲核试剂影响DNA中特定磷酸二酯键的裂解。我们使用过氧化氢与酿酒酵母的Flp重组酶一起进行该反应，该重组酶激活磷酸二酯以供可扩散的小亲核试剂攻击。虽然Flp能够自行切割DNA，但我们报告的反应发生在正常情况下不会被酶切割的Flp结合位点。此外，过氧化物介导的DNA骨架的切割用缺乏活性位点酪氨酸的Flp突变体发生，所述活性位点酪氨酸在野生型Flp中攻击DNA骨架以形成共价脱氧核糖-3 '-磷酸酪氨酰中间体（Evansetal.，1990; Gronostajski&Sadowski，1985）。除了提供新的信息的途径，DNA裂解的Flp，我们在这里展示了如何构建一个试剂，使一个单一的，特定的切口在一条链的DNA双链体。
Hydrogen peroxide is capable of effecting thecleavage of a specific phosphodiester bond in DNA, when used in concert with the recombinase enzyme Flp from Saccharomyces cerevisiae. This cleavage is not caused by oxidative damage of the DNA backbone but instead is the result of nucleophilic attack by peroxide. A single phosphorus-oxygen bond is broken in the reaction. Cleavage of DNA by peroxide also occurs with an inactive mutant of Flp in which the activesite nucleophile tyrosine has been replaced by phenylalanine. Besides providing information on the mechanism of strand cleavage by Flp, these results may contribute to the development of new synthetic DNA cleavage reagents that act by hydrolytic and not radical chemistry.The phosphodiester backbone of DNA is notoriously resistant to hydrolytic cleavage. As a consequence, many small molecules that are used to degrade DNA cleave the deoxyribose backbone via radical chemistry (Stubbe & Kozarich, 1987). Enzymes, of course, are capable of efficiently hydrolyzing the phosphodiester bonds of DNA. We report here a system in which hydrogen peroxide acts as a nucleophile to effect the cleavage of a specific phosphodiester bond in DNA. We perform this reaction using hydrogen peroxide in concert with the Flp recombinase of Saccharomyces cerevisae, which activates the phosphodiester for attack by the diffusible small nucleophile. While Flp is capable of cleaving DNA on its own, the reaction we report occurs at a Flp binding site that is not cut by the enzyme under normal circumstances. Furthermore, peroxide-mediated cleavage of the DNA backbone takes place with a Flp mutant that lacks the active site tyrosine which in wild-type Flp attacks the DNA backbone to form a covalent deoxyribose-3'-phosphotyrosyl intermediate (Evansetal., 1990; Gronostajski&Sadowski, 1985). Besides providing new information on the pathway of DNA cleavage by Flp, we show here how to construct a reagent which makes a single, specific nick in one strand of a DNA duplex.