Yarnell A T, Oh S, Reinberg D, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
J Biol Chem. 2001 Jul 13;276(28):25736-41. doi: 10.1074/jbc.M101208200. Epub 2001 May 8.
The structure-specific recognition protein SSRP1, initially isolated from expression screening of a human B-cell cDNA library for proteins that bind to cisplatin (cis-diamminedichloroplatinum(II))-modified DNA, contains a single DNA-binding high mobility group (HMG) domain. Human SSRP1 purifies as a heterodimer of SSRP1 and Spt16 (FACT) that alleviates the nucleosomal block to transcription elongation by RNAPII in vitro. The affinity and specificity of FACT, SSRP1, and the isolated HMG domain of SSRP1 for cisplatin-damaged DNA were investigated by gel mobility shift assays. FACT exhibits both affinity and specificity for DNA damaged globally with cisplatin compared with unmodified DNA or DNA damaged globally with the clinically ineffective trans-DDP isomer. FACT binds the major 1,2-d(GpG) intrastrand cisplatin adduct, but its isolated SSRP1 subunit fails to form discrete, high affinity complexes with cisplatin-modified DNA under similar conditions. These results suggest that Spt16 primes SSRP1 for cisplatin-damaged DNA recognition by unveiling its HMG domain. As expected, the isolated HMG domain of SSRP1 is sufficient for specific binding to cisplatin-damaged DNA and binds the major cisplatin 1,2-d(GpG) intrastrand cross-link. The affinity and specificity of FACT for cisplatin-modified DNA, as well as its importance for transcription of chromatin, suggests that the interaction of FACT and cisplatin-damaged DNA may be crucial to the anticancer mechanism of cisplatin.
结构特异性识别蛋白SSRP1最初是通过对人B细胞cDNA文库进行表达筛选而分离得到的，该文库用于筛选与顺铂（顺二氯二氨合铂（II））修饰的DNA结合的蛋白质，它包含一个单一的DNA结合高迁移率族（HMG）结构域。人SSRP1以SSRP1和Spt16（FACT）的异二聚体形式纯化，该异二聚体在体外可减轻核小体对RNA聚合酶II转录延伸的阻碍。通过凝胶迁移率变动分析研究了FACT、SSRP1以及SSRP1分离出的HMG结构域对顺铂损伤DNA的亲和力和特异性。与未修饰的DNA或用临床上无效的反式-DDP异构体整体损伤的DNA相比，FACT对用顺铂整体损伤的DNA表现出亲和力和特异性。FACT结合主要的1,2-二（GpG）链内顺铂加合物，但在类似条件下，其分离出的SSRP1亚基无法与顺铂修饰的DNA形成离散的高亲和力复合物。这些结果表明，Spt16通过揭示其HMG结构域，使SSRP1能够识别顺铂损伤的DNA。正如预期的那样，SSRP1分离出的HMG结构域足以特异性结合顺铂损伤的DNA，并结合主要的顺铂1,2-二（GpG）链内交联。FACT对顺铂修饰的DNA的亲和力和特异性，以及其对染色质转录的重要性，表明FACT与顺铂损伤的DNA之间的相互作用可能对顺铂的抗癌机制至关重要。
