Zamble D B, Mu D, Reardon J T, Sancar A, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139, USA.
Biochemistry. 1996 Aug 6;35(31):10004-13. doi: 10.1021/bi960453+.
Nucleotide excision repair is one of the many cellular defense mechanisms against the toxic effects of cisplatin. An in vitro excision repair assay employing mammalian cell-free extracts was used to determine that the 1,2-d(ApG) intrastrand cross-link, a prevalent cisplatin-DNA adduct, is excised by the excinuclease from a site-specifically modified oligonucleotide 156 base pairs in length. Repair of the minor interstrand d(G)/d(G) cross-link was not detected by using this system. Proteins containing the high mobility group (HMG) domain DNA-binding motif, in particular, rat HMG1 and a murine testis-specific HMG-domain protein, specifically inhibit excision repair of the intrastrand 1,2-d(GpG) and -d(ApG) cross-links. This effect was also exhibited by a single HMG domain from HMG1. Similar inhibition of repair of a site-specific 1,2-d(GpG) intrastrand cross-link by an HMG-domain protein also occurred in a reconstituted system containing highly purified repair factors. These results indicate that HMG-domain proteins can block excision repair of the major cisplatin-DNA adducts and suggest that such an activity could contribute to the unique sensitivity of certain tumors to the drug. The reconstituted excinuclease was more efficient at excising the 1,3-d(GpTpG) intrastrand adduct than either the 1,2-d(GpG) or d(ApG) intrastrand adducts, in agreement with previous experiments using whole cell extracts [Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394-10398]. This result suggests that structural differences among the platinated DNA substrates, and not the presence of unidentified cellular factors, determine the relative excision repair rates of cisplatin-DNA intrastrand cross-links in the whole cell extracts.
核苷酸切除修复是众多细胞防御机制之一，可抵御顺铂的毒性作用。采用哺乳动物无细胞提取物的体外切除修复试验确定，1,2 - d(ApG)链内交联物（一种常见的顺铂 - DNA加合物）可被核酸外切酶从一个长度为156个碱基对的位点特异性修饰寡核苷酸上切除。使用该系统未检测到较小的链间d(G)/d(G)交联物的修复情况。含有高迁移率族（HMG）结构域DNA结合基序的蛋白质，特别是大鼠HMG1和一种小鼠睾丸特异性HMG结构域蛋白，可特异性抑制1,2 - d(GpG)和 - d(ApG)链内交联物的切除修复。HMG1的单个HMG结构域也表现出这种效应。在含有高度纯化修复因子的重组系统中，HMG结构域蛋白对位点特异性1,2 - d(GpG)链内交联物修复的类似抑制作用也会发生。这些结果表明，HMG结构域蛋白可阻断主要顺铂 - DNA加合物的切除修复，并提示这种活性可能导致某些肿瘤对该药物具有独特的敏感性。与之前使用全细胞提取物的实验一致[Huang, J. -C., Zamble, D. B., Reardon, J. T., Lippard, S. J., Sancar, A. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10394 - 10398]，重组核酸外切酶切除1,3 - d(GpTpG)链内加合物的效率高于1,2 - d(GpG)或d(ApG)链内加合物。这一结果表明，铂化DNA底物之间的结构差异而非未鉴定细胞因子的存在，决定了全细胞提取物中顺铂 - DNA链内交联物的相对切除修复率。
