Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity
Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity
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DOI：
10.1124/mol.56.6.1105
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发表时间：
1999-12-01
影响因子：
3.6
通讯作者：
Benedetti, P
中科院分区：
文献类型：
--
作者：
Fiorani, P;Amatruda, JF;Benedetti, P
DNA拓扑异构酶I（Top1p）通过形成共价中间体来松弛超螺旋DNA，其中活性位点酪氨酸瞬时结合到切断的DNA链。抗肿瘤剂喜树碱（Cpt）特异性靶向Top1p，并且已经分离出使酶Cpt耐药的几种突变。突变的残基，虽然位于酶的不同区域，可能构成Cpt结合位点的一部分。为了开始确定DNA Top1p的结构特征对Cpt诱导的细胞毒性很重要，我们开发了一种新的酵母遗传筛选，从人类Top1突变体中分离出具有催化活性但耐Cpt的酶。在分离的突变中，Ser或瓦尔取代Gly363，这与我们以前报道的Gly363到Cys的突变一样，抑制了Top1p的Cpt敏感性。相比之下，每个氨基酸取代不同的能力，以抑制致命的表型和催化活性的人top1突变体top1T718A类似Cpt通过稳定的共价中间体。生化分析和分子建模支持的模型，其中两个保守的结构域，一个中心的“唇”区域包含残基Gly363和周围的活性位点酪氨酸（Tyr723）的残基之间的相互作用，直接影响Cpt结合位点和酶催化的形成。
DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand. The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant. The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site. To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants. Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p. In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate. Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.