Treiber D K, Zhai X, Jantzen H M, Essigmann J M
Department of Chemistry, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1994 Jun 7;91(12):5672-6. doi: 10.1073/pnas.91.12.5672.
The toxicity of DNA-damaging agents is widely believed to result from the formation of lesions that block polymerases or disrupt the integrity of the genome. A mechanism heretofore not addressed is that DNA damage may titrate essential DNA-binding proteins away from their natural sites of action. This report shows that the ribosomal RNA (rRNA) transcription factor hUBF (human upstream binding factor) binds with striking affinity (Kd(app) approximately 60 pM) to the intrastrand cis-Pt(NH3)2 crosslink formed by the anticancer drug cis-diamminedichloroplatinum(II) (cisplatin). When protein blots of human cell extracts are probed with cisplatin-modified DNA, 97- and 94-kDa proteins are detected, consistent with the known sites of hUBF species. A similar analysis of blots containing in vitro translated hUBF confirmed that the protein binds cisplatin adducts with high specificity. By contrast, DNA adducts of the clinically ineffective trans isomer of cisplatin, trans-diamminedichloroplatinum(II), are not recognized by hUBF. DNase I inhibition patterns of hUBF bound to a 100-base-pair DNA fragment containing a centrally located cis-Pt(NH3)2-d(GpG) crosslink reveal specific protein-DNA interactions in a 14-base-pair region flanking the adduct. The affinity of hUBF for the rRNA promoter is similar (Kd(app) approximately 18 pM) to that measured for the cisplatin adduct. In addition, we observe that the hUBF-promoter interaction is highly sensitive to the antagonistic effects of cisplatin-DNA adducts. These results suggest that a cisplatin-mediated transcription-factor-hijacking mechanisms could disrupt rRNA synthesis, which is stimulated in proliferating cells.
人们普遍认为，DNA损伤剂的毒性源于形成的损伤会阻碍聚合酶或破坏基因组的完整性。一种迄今未被探讨的机制是，DNA损伤可能会使重要的DNA结合蛋白从其天然作用位点上被滴定下来。本报告表明，核糖体RNA（rRNA）转录因子hUBF（人类上游结合因子）与抗癌药物顺二氯二氨铂（II）（顺铂）形成的链内顺式-Pt(NH3)2-d(GpG)交联具有惊人的亲和力（Kd(app)约为60 pM）。当用顺铂修饰的DNA探测人类细胞提取物的蛋白质印迹时，检测到97 kDa和94 kDa的蛋白质，这与已知的hUBF种类位点一致。对含有体外翻译的hUBF的印迹进行类似分析，证实该蛋白与顺铂加合物具有高度特异性结合。相比之下，顺铂的临床无效反式异构体反二氯二氨铂（II）的DNA加合物不被hUBF识别。与含有位于中心位置的顺式-Pt(NH3)2-d(GpG)交联的100个碱基对DNA片段结合的hUBF的DNase I抑制模式揭示了加合物两侧14个碱基对区域内特定的蛋白质-DNA相互作用。hUBF对rRNA启动子的亲和力与对顺铂加合物测得的亲和力相似（Kd(app)约为18 pM）。此外，我们观察到hUBF-启动子相互作用对顺铂-DNA加合物的拮抗作用高度敏感。这些结果表明，顺铂介导的转录因子劫持机制可能会破坏rRNA合成，而rRNA合成在增殖细胞中会受到刺激。
