Binding of the transcription factor, Sp1, to non-target sites in DNA modified by benzo[a]pyrene diol epoxide.
Binding of the transcription factor, Sp1, to non-target sites in DNA modified by benzo[a]pyrene diol epoxide.
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转录因子 Sp1 与苯并[a]芘二醇环氧化物修饰的 DNA 中非靶位点的结合。
DOI：
10.1093/carcin/16.5.975
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发表时间：
1995
期刊：
影响因子：
4.7
通讯作者：
Tran,N
中科院分区：
文献类型：
--
作者：
MacLeod,MC;Powell,KL;Tran,N
致癌物7 r，8 t-dihydroxy-9 t，10 t-oxy-7，8，9，10-tetrahydrobenzo[a]pyrene（BPDE）与DNA的共价结合导致加合物位点周围DNA构象的变化。然而，这种致癌物-DNA加合物对DNA与特定蛋白质相互作用的影响很少受到关注。转录因子Spl与仓鼠腺苷磷酸核糖转移酶基因启动子中的GC盒序列的结合是一个有用的模型系统。电泳迁移率变动分析，竞争实验和DNase I足迹证明亲和纯化，人Sp1的启动子片段中的两个相邻的GC盒的特异性结合。出乎意料的是，用BPDE将该DNA片段修饰到高水平（约7%的核苷酸），导致Sp1的表观亲和力显著增加（5至10倍）。不含GC盒的异源DNA片段不竞争Sp1与启动子的结合，除非它先前用BPDE修饰。此外，两个不含GC盒的DNA片段仅在BPDE修饰时才表现出Sp1依赖的迁移率变化。BPDE修饰的Sp1结合启动子片段的DNA酶I足迹没有显示出特定的结合位点，这表明许多BPDE-DNA加合物位点可以与蛋白质相互作用。一个模型中，Sp1结合到非靶位点的静态弯曲或诱导的灵活性在加合物的网站增强进行了讨论。
Covalent binding of the carcinogen, 7r,8t-dihydroxy-9t,10t-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), to DNA causes changes in the conformation of the DNA around the site of the adduct. However, the influence of such carcinogen-DNA adducts on interactions of the DNA with specific proteins has received little attention. Binding of the transcription factor, Spl, to GC-box sequences in the promoter of the hamster adenosine phosphoribosyl transferase gene is a useful model system. Electrophoretic mobility shift assays, competition experiments and DNase I footprinting demonstrated specific binding of affinitypurified, human Sp1 to two adjacent GC-boxes in the promoter fragment. Unexpectedly, modification of this DNA fragment to high levels (∼7% of the nucleotides) with BPDE caused a substantial (5- to 10-fold) increase in the apparent affinity of Sp1. A heterologous DNA fragment that contained no GC-boxes did not compete for the binding of Sp1 to the promoter, unless it was previously modified with BPDE. In addition, two DNA fragments that contained no GC-boxes exhibited Sp1-dependent mobility shifts only when modified by BPDE. DNase I footprinting of the BPDE-modified, Sp1-bound promoter fragment did not reveal specific sites of binding, suggesting that numerous BPDE-DNA adduct sites can interact with the protein. A model in which Sp1 binding to non-target sites is enhanced by a static bend or an induced flexibility at the site of an adduct is discussed.