Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease.
Replacement of the active site tyrosine of vaccinia DNA topoisomerase by glutamate, cysteine or histidine converts the enzyme into a site-specific endonuclease.
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用谷氨酸、半胱氨酸或组氨酸取代痘苗 DNA 拓扑异构酶的活性位点酪氨酸，将该酶转化为位点特异性核酸内切酶。
DOI：
10.1093/nar/26.2.490
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发表时间：
1998
影响因子：
14.9
通讯作者：
Shuman,S
中科院分区：
文献类型：
--
作者：
Wittschieben,J;Petersen,BO;Shuman,S
牛痘拓扑异构酶在双链DNA的5′-CCCTT ↓位点形成共价蛋白-DNA中间体。T ↓核苷酸通过3′-磷酸二酯键与酶的Tyr-274连接。在这里，我们报告了含有谷氨酸、半胱氨酸或组氨酸代替Tyr-274的突变酶催化CCCTT ↓位点处60 bp双链DNA的核酸内切裂解，产生3′磷酸终止产物。Cys-274突变体形成痕量水平的共价蛋白-DNA复合物，表明DNA切割反应可能通过半胱氨酰-磷酸中间体进行。然而，His-274和Glu-274突变体显示没有可检测到的共价蛋白质-DNA加合物的积累。Glu-274是所测试的突变体中活性最高的。Glu-274的内切核酸酶活性的pH依赖性（最适pH = 6.5）不同于野生型酶在共价加合物的水解中的pH依赖性（最适pH = 9.5）。在pH 6.5下，Glu-274内切核酸酶反应比野生型拓扑异构酶形成共价加合物的速率慢5-6个数量级，但比野生型共价加合物水解的速率快1020倍。我们讨论了两个潜在的机制来解释拓扑异构酶到核酸内切酶的明显转换。
Vaccinia topoisomerase forms a covalent protein-DNA intermediate at 5′-CCCTT ↓ sites in duplex DNA. The T ↓ nucleotide is linked via a 3′-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that mutant enzymes containing glutamate, cysteine or histidine in lieu of Tyr-274 catalyze endonucleolytic cleavage of a 60 bp duplex DNA at the CCCTT ↓ site to yield a 3′ phosphate-terminated product. The Cys-274 mutant forms trace levels of a covalent protein-DNA complex, suggesting that the DNA cleavage reaction may proceed through a cysteinyl-phosphate intermediate. However, the His-274 and Glu-274 mutants evince no detectable accumulation of a covalent protein-DNA adduct. Glu-274 is the most active of the mutants tested. The pH dependence of the endonuclease activity of Glu-274 (optimum pH = 6.5) is distinct from that of the wild-type enzyme in hydrolysis of the covalent adduct (optimum pH = 9.5). At pH 6.5, the Glu-274 endonuclease reaction is slower by 5–6 orders of magnitude than the rate of covalent adduct formation by the wild-type topoisomerase, but is ∼20 times faster than the rate of hydrolysis by the wild-type covalent adduct. We discuss two potential mechanisms to account for the apparent conversion of a topoisomerase into an endonuclease.