The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis
The interaction profile of homologous recombination repair proteins RAD51C, RAD51D and XRCC2 as determined by proteomic analysis
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DOI：
10.1002/pmic.200800977
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发表时间：
2009-08-01
期刊：
影响因子：
3.4
通讯作者：
Pittman, Douglas L.
中科院分区：
文献类型：
--
作者：
Rajesh, Changanamkandath;Gruver, Aaron M.;Pittman, Douglas L.
RAD 51蛋白家族参与同源重组（HR）DNA修复和维持染色体完整性。为了鉴定与HR蛋白相互作用的候选物，使用细菌表达系统纯化小鼠RAD 51 C、RAD 51 D和XRCC 2蛋白，并且它们中的每一个用于从小鼠胚胎成纤维细胞提取物共沉淀相互作用伴侣。对丝裂霉素C处理和未处理的小鼠胚胎成纤维细胞的细胞提取物的共沉淀洗脱液的1-D SDS-PAGE后获得的蛋白条带进行质谱分析。相互作用蛋白质的分析显示出对核酸结合和修饰蛋白质的明显偏好。通过共沉淀洗脱液的Western印迹分析证实了四种候选蛋白（SFPQ，NONO，MSH 2和微型染色体维持蛋白2）的相互作用，并且还验证了与RAD 51 D形成离体复合物。其他相互作用的蛋白质与细胞分裂，胚胎发育，蛋白质和碳水化合物代谢，细胞运输，蛋白质合成，修饰或折叠，细胞结构或运动功能。这项研究的结果是确定RAD 51旁系同源物相互作用伙伴和了解帮助或调节FIR修复机制的蛋白质功能多样性的重要一步。
The RAD51 family of proteins is involved in homologous recombination (HR) DNA repair and maintaining chromosome integrity. To identify candidates that interact with HR proteins, the mouse RAD51C, RAD51D and XRCC2 proteins were purified using bacterial expression systems and each of them used to co-precipitate interacting partners from mouse embryonic fibroblast cellular extracts. Mass spectroscopic analysis was performed on protein bands obtained after 1-D SDS-PAGE of co-precipitation eluates from cell extracts of mitomycin C treated and untreated mouse embryonic fibroblasts. Profiling of the interacting proteins showed a clear bias toward nucleic acid binding and modification proteins. Interactions of four candidate proteins (SFPQ, NONO, MSH2 and mini chromosome maintenance protein 2) were confirmed by Western blot analysis of co-precipitation eluates and were also verified to form ex vivo complexes with RAD51D. Additional interacting proteins were associated with cell division, embryo development, protein and carbohydrate metabolism, cellular trafficking, protein synthesis, modification or folding, and cell structure or motility functions. Results from this study are an important step toward identifying interacting partners of the RAD51 paralogs and understanding the functional diversity of proteins that assist or regulate FIR repair mechanisms.