Matsunaga T, Park C H, Bessho T, Mu D, Sancar A
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill 27599, USA.
J Biol Chem. 1996 May 10;271(19):11047-50. doi: 10.1074/jbc.271.19.11047.
XPF-ERCC1 and XPG proteins are nucleases that are involved in human nucleotide excision repair. In this study, we characterized the structure-specific junction-cutting activities of both nucleases using DNA substrates containing a bubble or loop structure. We found that the junction-cutting activities of XPF-ERCC1 and XPG were greatly stimulated by human replication protein A (RPA), while heterologous single-stranded DNA-binding proteins could not substitute for human RPA. To test for specific interaction between RPA and XPF-ERCC1 as is known to occur between RPA and XPG, we employed a pull-down assay with immobilized "bubble" substrate. We found that the binding of XPF-ERCC1 complex to the bubble substrate was enhanced by RPA, suggesting a possible mechanism for RPA in the excision nuclease system, that is the targeting of the nuclease subunits to their specific sites of action. Furthermore, the RPA-promoted junction cutting by XPF-ERCC1 and XPG nucleases was observed with "loop" substrates as well, raising the possibility that XPF-ERCC1, XPG, and RPA may function in removing loop structures from DNA, independent of the other subunits of the human excinuclease.
XPF-ERCC1和XPG蛋白是参与人类核苷酸切除修复的核酸酶。在本研究中，我们使用含有气泡或环结构的DNA底物来表征这两种核酸酶的结构特异性连接切割活性。我们发现，XPF-ERCC1和XPG的连接切割活性受到人类复制蛋白A（RPA）的极大刺激，而异源单链DNA结合蛋白不能替代人类RPA。为了测试RPA与XPF-ERCC1之间是否像已知的RPA与XPG之间那样存在特异性相互作用，我们采用了固定化“气泡”底物的下拉实验。我们发现，RPA增强了XPF-ERCC1复合物与气泡底物的结合，这表明RPA在切除核酸酶系统中的一种可能机制，即核酸酶亚基靶向其特定作用位点。此外，在“环”底物上也观察到了RPA促进的XPF-ERCC1和XPG核酸酶的连接切割，这增加了XPF-ERCC1、XPG和RPA可能独立于人类外切核酸酶的其他亚基从DNA中去除环结构的可能性。
