Chow C S, Barnes C M, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Biochemistry. 1995 Mar 7;34(9):2956-64. doi: 10.1021/bi00009a027.
Proteins containing a relatively new DNA-binding motif known as the high-mobility group (HMG) domain bind specifically to DNA modified by the anticancer drug cisplatin, but not to unmodified DNA (McA'Nulty & Lippard, 1995). Southwestern-blot analyses of the binding of proteolytic fragments of HMG1 to a 123-bp globally platinated DNA demonstrate that the HMG domains A and B of HMG1 are responsible for its specific interactions with cisplatin-modified DNA. An 81 amino acid recombinant protein representing a single HMG motif, HMG1 domain B, binds with an affinity (Kd = 10(-7) M) equal to that of HMG1 itself to 92- and 100-bp DNAs containing the major adduct of cisplatin, a cis-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]] intrastrand cross-link, at a specific site. The isolated HMG domain B binds with comparable affinity to cisplatin-modified DNAs having as few as 20 bp. The related human mitochondrial HMG domain protein mtTFA also recognizes the 123-bp globally platinated DNA, providing further evidence that HMG domains are responsible for modulating binding of this class of proteins to cisplatin-modified DNA. This work provides direct biochemical evidence in support of conclusions drawn previously from analyses of sequence conservation (Bruhn et al., 1992) that HMG domains are the key elements in protein binding to cisplatin-modified DNA.
含有一种相对较新的被称为高迁移率族（HMG）结构域的DNA结合基序的蛋白质，能特异性结合由抗癌药物顺铂修饰的DNA，但不能结合未修饰的DNA（麦克纳尔蒂和利帕德，1995年）。对HMG1蛋白水解片段与123bp全铂化DNA结合的蛋白质印迹分析表明，HMG1的HMG结构域A和B负责其与顺铂修饰DNA的特异性相互作用。一种代表单个HMG基序的81个氨基酸的重组蛋白，即HMG1结构域B，以与HMG1本身相同的亲和力（Kd = 10^(-7) M）在特定位点结合含有顺铂主要加合物（顺式-[Pt(NH3)2-[d(GpG)-N7(1), -N7(2)]]链内交联）的92bp和100bp DNA。分离出的HMG结构域B以相当的亲和力结合至少20bp的顺铂修饰DNA。相关的人类线粒体HMG结构域蛋白mtTFA也能识别123bp全铂化DNA，这进一步证明HMG结构域负责调节这类蛋白质与顺铂修饰DNA的结合。这项工作提供了直接的生化证据，支持先前从序列保守性分析得出的结论（布鲁恩等人，1992年），即HMG结构域是蛋白质结合顺铂修饰DNA的关键元件。
