Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro.
Human replication protein A preferentially binds cisplatin-damaged duplex DNA in vitro.
复制标题
人复制蛋白 A 在体外优先结合顺铂损伤的双链 DNA。
DOI：
10.1021/bi9730590
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Turchi,JJ
中科院分区：
文献类型：
--
作者：
Patrick,SM;Turchi,JJ
通过顺铂- DNA亲和层析分离人细胞提取物，鉴定能够结合顺铂损伤DNA的蛋白质。一个特定的蛋白质- DNA复合物，称为DRP-3，在电泳迁移迁移试验（EMSA）中鉴定使用顺铂损伤DNA探针。通过这个实验，我们纯化了DRP-3，最终的部分含有70、53、46、32和14 kDa的蛋白质。根据亚基分子量、抗体反应性和DNA结合活性，DRP-3被鉴定为人复制蛋白A （human replication protein A, hRPA）。因此，我们在体外评估了重组人RPA （rhRPA）与双顺铂损伤DNA的结合。用顺铂处理高度纯化的完全双工44-bp DNA，与未损伤的对照相比，rhRPA结合增加了10 - 20倍。对RPA - DNA复合物的稳定性进行了评估，NaCl和mgcl2浓度完全抑制了rhRPA与未损伤DNA的结合，但对双铂DNA的结合影响很小。我们评估了rhRPA与含有单位点特异性1,2-d（GpG）顺铂加合物的双工DNA的结合，结果显示与相同序列的未受损对照DNA相比，rhRPA与该DNA底物的结合增加了4 - 6倍。这些结果与RPA参与对顺铂损伤DNA的初始识别一致，可能介导DNA修复事件。因此，我们评估了另一种顺铂DNA结合蛋白HMG-1如何影响rhRPA结合受损DNA的能力。竞争结合分析显示，在反应过程中，任何一种蛋白质与顺铂损伤的DNA的解离都很小。同时加入实验发现，rhRPA对HMG-1与顺铂损伤DNA结合的影响最小，而HMG-1抑制了rhRPA与损伤DNA的结合活性。这些数据与HMG-1阻断DNA修复一致，可能具有增强顺铂药物细胞毒性的能力。
Fractionation of human cell extracts by cisplatin−DNA affinity chromatography was employed to identify proteins capable of binding cisplatin-damaged DNA. A specific protein−DNA complex, termed DRP-3, was identified in an electrophoretic mobility shift assay (EMSA) using a cisplatin-damaged DNA probe. Using this assay we purified DRP-3 and the final fraction contained proteins of 70, 53, 46, 32, and 14 kDa. On the basis of subunit molecular weights, antibody reactivity, and DNA binding activities, DRP-3 was identified as human replication protein A (hRPA). Therefore, we assessed the binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA in vitro. Global treatment of a highly purified completely duplex 44-bp DNA with cisplatin resulted in a 10−20-fold increase in rhRPA binding compared to the undamaged control. The stability of the RPA−DNA complexes was assessed, and NaCl and MgCl2concentrations that completely inhibited rhRPA binding to undamaged DNA had only a minimal effect on binding to duplex platinated DNA. We assessed rhRPA binding to a duplex DNA containing a single site-specific 1,2-d(GpG) cisplatin adduct, and the results revealed a 4−6-fold increase in binding to this DNA substrate compared to an undamaged control DNA of identical sequence. These results are consistent with RPA being involved in the initial recognition of cisplatin-damaged DNA, possibly mediating DNA repair events. Therefore, we assessed how another cisplatin DNA binding protein, HMG-1, affected the ability of rhRPA to bind damaged DNA. Competition binding assays show minimal dissociation of either protein from cisplatin-damaged DNA during the course of the reaction. Simultaneous addition experiments revealed that HMG-1 binding to cisplatin-damaged DNA was minimally affected by rhRPA, while HMG-1 inhibited the damaged-DNA binding activity of rhRPA. These data are consistent with HMG-1 blocking DNA repair and possibly having the capability to enhance the cytotoxic efficacy of the drug cisplatin.