Wei M, Cohen S M, Silverman A P, Lippard S J
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
J Biol Chem. 2001 Oct 19;276(42):38774-80. doi: 10.1074/jbc.M106374200. Epub 2001 Aug 20.
The results presented describe the effects of various spectator ligands, attached to a platinum 1,2-intrastand d(GpG) cross-link in duplex DNA, on the binding of high mobility group box (HMGB) domains and the TATA-binding protein (TBP). In addition to cisplatin-modified DNA, 15-base pair DNA probes modified by Pt(1R,2R-diaminocyclohexane), cis-Pt(NH(3))(cyclohexylamine), Pt(ethylenediamine), cis-Pt(NH(3))(cyclobutylamine), and cis-Pt(NH(3))(2-picoline) were examined. Electrophoretic mobility shift assays show that both the A and B domains of HMGB1 as well as TBP discriminate between different platinum-DNA adducts. HMGB1 domain A is the most sensitive to the nature of the spectator ligands on platinum. The effect of the spectator ligands on protein binding also depends highly on the base pairs flanking the platinated d(GpG) site. Double-stranded oligonucleotides containing the AGGC sequence, where the asterisks denote the sites of platination, with different spectator ligands are only moderately discriminated by the HMGB proteins and TBP, but the recognition of dsTGGA is highly dependent on the ligands. The effects of HMGB1 overexpression in a BG-1 ovarian cancer cell line, induced by steroid hormones, on the sensitivity of cells treated with [Pt(1R,2R-diaminocyclohexane)Cl(2)] and cis-[Pt(NH(3))(cyclohexylamine)Cl(2)] were also examined. The results suggest that HMGB1 protein levels influence the cellular processing of cis-Pt(NH(3))- (cyclohexylamine), but not Pt((1R,2R)-diaminocyclohexane), DNA lesions. This result is consistent with the observed binding of HMGB1a to platinum-modified dsTGGA probes but not with the binding affinity of HMGB1a and HMGB1 to platinum-damaged dsAGGC oligonucleotides. These experiments reinforce the importance of sequence context in platinum-DNA lesion recognition by cellular proteins.
所呈现的结果描述了连接在双链DNA中铂1,2-链内d(GpG)交联上的各种旁观配体对高迁移率族框（HMGB）结构域和TATA结合蛋白（TBP）结合的影响。除了顺铂修饰的DNA外，还检测了用Pt(1R,2R-二氨基环己烷)、顺式-Pt(NH(3))(环己胺)、Pt(乙二胺)、顺式-Pt(NH(3))(环丁胺)和顺式-Pt(NH(3))(2-甲基吡啶)修饰的15碱基对DNA探针。电泳迁移率变动分析表明，HMGB1的A和B结构域以及TBP能够区分不同的铂-DNA加合物。HMGB1结构域A对铂上旁观配体的性质最为敏感。旁观配体对蛋白质结合的影响还高度依赖于铂化d(GpG)位点两侧的碱基对。含有AGGC序列（星号表示铂化位点）且带有不同旁观配体的双链寡核苷酸仅被HMGB蛋白和TBP适度区分，但dsTGGA的识别高度依赖于配体。还检测了类固醇激素诱导的BG-1卵巢癌细胞系中HMGB1过表达对用[Pt(1R,2R-二氨基环己烷)Cl(2)]和顺式-[Pt(NH(3))(环己胺)Cl(2)]处理的细胞敏感性的影响。结果表明，HMGB1蛋白水平影响顺式-Pt(NH(3))-(环己胺)的细胞处理过程，但不影响Pt((1R,2R)-二氨基环己烷)的DNA损伤处理过程。这一结果与观察到的HMGB1a与铂修饰的dsTGGA探针的结合一致，但与HMGB1a和HMGB1与铂损伤的dsAGGC寡核苷酸的结合亲和力不一致。这些实验强化了序列背景在细胞蛋白识别铂-DNA损伤中的重要性。
