Nature of full-length HMGB1 binding to cisplatin-modified DNA
Nature of full-length HMGB1 binding to cisplatin-modified DNA
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DOI：
10.1021/bi026972w
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发表时间：
2003-03-11
期刊：
影响因子：
2.9
通讯作者：
Lippard, SJ
中科院分区：
文献类型：
--
作者：
Jung, YW;Lippard, SJ
HMGB1是一种高度保守的非组蛋白DNA结合蛋白，与特定的DNA结构基序相互作用，如在顺铂损伤、四向连接和超级线圈中遇到的DNA结构基序。采用羟基自由基足迹法和电泳凝胶迁移位移法研究了全长HMGB1与顺铂修饰DNA的相互作用。HMGB1含有两个串联HMG盒结构域和一个c端酸性尾部。全长HMGB1蛋白主要通过结构域a与含有1,2-链内d（GpG）交联的DNA结合，解离常数K-d为120 nM。全长HMGB1中HMG结构域A和B插入残基的位点定向突变进一步支持了只有一个HMG盒子结构域与顺铂损伤位点结合的结论。通过EDC交联实验检测了c端尾部与HMGB1蛋白其余部分的相互作用。在HMGB1中，酸尾主要与结构域B和连接子区相互作用，而不是与结构域A相互作用。这些结果阐明了串联HMG盒子和HMGB1的c端酸尾在结合DNA和抗癌药物顺铂形成的主要DNA加合物中的各自作用。
HMGB1, a highly conserved non-histone DNA-binding protein, interacts with specific DNA structural motifs such as those encountered at cisplatin damage, four-way junctions, and supercoils. The interaction of full-length HMGB1, containing two tandem HMG box domains and a C-terminal acidic tail, with cisplatin-modified DNA was investigated by hydroxyl radical footprinting and electrophoretic gel mobility shift assays. The full-length HMGB1 protein binds to DNA containing a 1,2-intrastrand d(GpG) cross-link mainly through domain A, as revealed by footprinting, with a dissociation constant K-d of 120 nM. Site-directed mutagenesis of intercalating residues in both HMG domains A and B in full-length HMGB1 further supports the conclusion that only one HMG box domain is bound to the site of cisplatin damage. Interaction of the C-terminal tail with the rest of the HMGB1 protein was examined by EDC cross-linking experiments. The acidic tail mainly interacts with domain B and linker regions rather than domain A in HMGB1. These results illuminate the respective roles of the tandem HMG boxes and the C-terminal acidic tail of HMGB1 in binding to DNA and to the major DNA adducts formed by the anticancer drug cisplatin.