Ohndorf U M, Whitehead J P, Raju N L, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Biochemistry. 1997 Dec 2;36(48):14807-15. doi: 10.1021/bi9717643.
The anticancer drug cisplatin is particularly effective against testicular tumors. Although the clinical consequences of cisplatin chemotherapy are well-known, the precise mechanism of action remains elusive. Specific recognition of cisplatin-damaged DNA by a class of proteins containing the high-mobility group (HMG) domain DNA-binding motif could play a role in mediating the cytotoxicity of the drug. This study presents a quantitative investigation of binding of the murine testis-specific high-mobility group protein tsHMG to DNA modified by cisplatin. The binding affinity and specificity of this protein to a site-specific 1,2-d(GpG) cisplatin-DNA intrastrand cross-link in a 20 bp probe were determined. A value for the apparent dissociation constant, Kd(app), of 24 +/- 5 nM was obtained by gel mobility shift assays. Binding competition assays with the corresponding unmodified 20 bp probe gave a ratio (rho) of nonspecific to specific Kd(app) values of 230. A polypeptide containing tsHMG domain A (residues 1-82) was expressed and purified to homogeneity. This domain alone was sufficient for specific recognition of cisplatin-modified DNA with a Kd(app) of 300 +/- 50 nM and a rho of 20, a comparatively high discrimination factor. DNase I interference analysis of the adduct-containing strand revealed that tsHMG binding extends over 14 nucleotides, centered around the platinated bases. The domain A polypeptide protection pattern covers a slightly smaller area of 13 nucleotides. The binding affinity and specificity of tsHMG for cisplatin-modified DNA are exceptional compared to those of other HMG-domain proteins studied previously. The possible relevance of these findings to the mechanism of action of cisplatin is discussed.
抗癌药物顺铂对睾丸肿瘤特别有效。尽管顺铂化疗的临床后果众所周知，但其确切作用机制仍不清楚。一类含有高迁移率族（HMG）结构域DNA结合基序的蛋白质对顺铂损伤的DNA的特异性识别可能在介导该药物的细胞毒性中起作用。本研究对小鼠睾丸特异性高迁移率族蛋白tsHMG与顺铂修饰的DNA的结合进行了定量研究。测定了该蛋白对20 bp探针中位点特异性1,2-d（GpG）顺铂-DNA链内交联的结合亲和力和特异性。通过凝胶迁移率变动分析获得了表观解离常数Kd（app）的值为24±5 nM。用相应的未修饰20 bp探针进行的结合竞争分析得出非特异性与特异性Kd（app）值的比值（rho）为230。表达并纯化了含有tsHMG结构域A（第1-82位氨基酸）的多肽，使其达到同质。仅该结构域就足以特异性识别顺铂修饰的DNA，Kd（app）为300±50 nM，rho为20，这是一个相对较高的区分因子。对含加合物链的DNase I干扰分析表明，tsHMG的结合延伸超过14个核苷酸，以铂化碱基为中心。结构域A多肽的保护模式覆盖了一个稍小的区域（覆盖13个核苷酸）。与先前研究的其他HMG结构域蛋白相比，tsHMG对顺铂修饰的DNA的结合亲和力和特异性非常突出。讨论了这些发现与顺铂作用机制的可能相关性。
