Patrick S M, Turchi J J
Department of Biochemistry and Molecular Biology, Wright State University School of Medicine, Dayton, Ohio 45435, USA.
J Biol Chem. 1999 May 21;274(21):14972-8. doi: 10.1074/jbc.274.21.14972.
Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.
复制蛋白A（RPA）是一种由70 kDa、34 kDa和14 kDa亚基组成的异源三聚体蛋白，已被证明是DNA复制、修复和同源重组所必需的。我们之前已经证明，与未受损的对照DNA相比，重组人RPA（rhRPA）与双链顺铂损伤的DNA具有优先结合作用（Patrick，S. M.，和Turchi，J. J.（1998年）《生物化学》37卷，8808 - 8815页）。在此，我们评估rhRPA与含有位点特异性顺铂 - DNA加合物的DNA的结合情况。结果表明，与含有1,2 - d(GpG)顺铂 - DNA链内加合物相比，rhRPA与含有单个1,3 - d(GpXpG)的30个碱基对双链底物的结合能力要强1.5至2倍，这与含有每种加合物的DNA的热稳定性差异一致。与这些数据相符的是，含有位于中心位置的单个链间顺铂交联的21个碱基对DNA底物与未受损对照DNA相比，结合较少。一系列测量rhRPA结合及同时进行的DNA变性实验表明，rhRPA通过产生单链DNA与双链顺铂损伤的DNA结合。单链DNA结合实验表明，与含有单个1,2 - d(GpG)顺铂 - DNA加合物的相同底物相比，rhRPA与未受损的24个碱基DNA的结合能力要强3至4倍。这些数据与rhRPA与双链损伤DNA的低亲和力相互作用一致，随后产生单链DNA，然后与未受损的DNA链进行高亲和力结合。
