DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair
DNA damage induced hyperphosphorylation of replication protein A. 2. Characterization of DNA binding activity, protein interactions, and activity in DNA replication and repair
复制标题
DOI：
10.1021/bi048057b
复制
发表时间：
2005-06-14
期刊：
影响因子：
2.9
通讯作者：
Turchi, JJ
中科院分区：
文献类型：
--
作者：
Patrick, SM;Oakley, GG;Turchi, JJ
复制蛋白A（RPA）是由70-、34-和14- kDa亚基组成的异源三聚体蛋白，其是包括DNA复制和DNA修复在内的许多DNA代谢过程所需的。使用在体外制备的纯化的高度磷酸化形式的RPA蛋白。我们已经讨论了过度磷酸化对稳态和前稳态DNA结合活性的影响，支持DNA修复和复制反应的能力，以及对与伴侣蛋白相互作用的影响。通过荧光偏振测量的平衡DNA结合活性揭示ssDNA与富含嘧啶的DNA序列结合没有差异。然而，RPA过度磷酸化导致其对富含嘌呤的ssDNA和双链体DNA底物的亲和力降低。预稳态动力学分析是一致的平衡DNA结合，并证明了从两个k（上）的贡献。和k（off）来实现这些差异。RPA的过度磷酸化形式保留了损伤特异性DNA结合，并且重要的是，过度磷酸化RPA对受损双链体DNA的亲和力是未修饰RPA对未受损双链体DNA的亲和力的3倍。超磷酸化RPA支持DNA修复的能力在支持核苷酸切除修复（NER）的能力方面显示出微小的差异。有趣的是，在反应条件下，RPA保持在一个高度磷酸化的形式。我们还观察到体外DNA复制的抑制。蛋白质-蛋白质相互作用的分析证实了过度磷酸化RPA对DNA代谢途径的影响。具体而言，RPA的磷酸化破坏了与DNA聚合酶α的相互作用，但对与XPA的相互作用没有显著影响。这些结果表明，DNA损伤诱导的RPA的过度磷酸化对DNA复制和DNA修复的影响是通过改变DNA结合活性和蛋白质-蛋白质相互作用介导的。
Replication protein A (RPA) is a heterotrimeric protein consisting of 70-, 34-, and 14- kDa subunits that is required for many DNA metabolic processes including DNA replication and DNA repair. Using a purified hyperphosphorylated form of RPA protein prepared in vitro. we have addressed file effects of hyperphosphorylation on steady-state and pre-steady-state DNA binding activity, the ability to support DNA repair and replication reactions, and the effect on the interaction with partner proteins. Equilibrium DNA binding activity measured by fluorescence polarization reveals no difference in ssDNA binding to pyrimidine-rich DNA sequences. However, RPA hyperphosphorylation results in it decreased affinity for purine-rich ssDNA and duplex DNA substrates. Pre-steady-state kinetic analysis is consistent with the equilibrium DNA binding and demonstrates a contribution from both the k(on). and k(off) to achieve these differences. The hyperphosphorylated form of RPA retains damage-specific DNA binding, and, importantly, the affinity of hyperphosphorylated RPA for damaged duplex DNA is 3-fold greater than the affinity of unmodified RPA for undamaged duplex DNA. The ability of hyperphosphorylated RPA to support DNA repair showed minor differences in the ability to support nucleotide excision repair (NER). Interestingly, under reaction conditions in which RPA is maintained in a hyperphosphorylated form. we also observed inhibition of in vitro DNA replication. Analyses of protein-protein interactions bear out the effects of hyperphosphorylated RPA on DNA metabolic pathways. Specifically, phosphorylation of RPA disrupts the interaction with DNA polymerase alpha but has no significant effect on the interaction with XPA. These results demonstrate that the effects of DNA damage induced hyperphosphorylation of RPA on DNA replication and DNA repair are mediated through alterations in DNA binding activity and protein-protein interactions.