Nishi Ryotaro, Alekseev Sergey, Dinant Christoffel, Hoogstraten Deborah, Houtsmuller Adriaan B, Hoeijmakers Jan H J, Vermeulen Wim, Hanaoka Fumio, Sugasawa Kaoru
Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Kobe, Hyogo 657-8501, Japan.
DNA Repair (Amst). 2009 Jun 4;8(6):767-76. doi: 10.1016/j.dnarep.2009.02.004. Epub 2009 Mar 21.
Although the basic principle of nucleotide excision repair (NER), which can eliminate various DNA lesions, have been dissected at the genetic, biochemical and cellular levels, the important in vivo regulation of the critical damage recognition step is poorly understood. Here we analyze the in vivo dynamics of the essential NER damage recognition factor XPC fused to the green fluorescence protein (GFP). Fluorescence recovery after photobleaching analysis revealed that the UV-induced transient immobilization of XPC, reflecting its actual engagement in NER, is regulated in a biphasic manner depending on the number of (6-4) photoproducts and titrated by the number of functional UV-DDB molecules. A similar biphasic UV-induced immobilization of TFIIH was observed using XPB-GFP. Surprisingly, subsequent integration of XPA into the NER complex appears to follow only the low UV dose immobilization of XPC. Our results indicate that when only a small number of (6-4) photoproducts are generated, the UV-DDB-dependent damage recognition pathway predominates over direct recognition by XPC, and they also suggest the presence of rate-limiting regulatory steps in NER prior to the assembly of XPA.
尽管核苷酸切除修复（NER）的基本原理，即能够消除各种DNA损伤，已在基因、生化和细胞水平上得到解析，但关键损伤识别步骤在体内的重要调控仍知之甚少。在此，我们分析了与绿色荧光蛋白（GFP）融合的必需NER损伤识别因子XPC的体内动态。光漂白后荧光恢复分析表明，紫外线诱导的XPC瞬时固定反映了其实际参与NER过程，其受双相调控方式的影响取决于（6-4）光产物的数量，并由功能性UV-DDB分子数量进行滴定。使用XPB-GFP观察到类似的紫外线诱导TFIIH双相固定。令人惊讶的是，随后XPA整合到NER复合物中似乎仅遵循XPC低紫外线剂量固定。我们的结果表明，当仅产生少量（6-4）光产物时，UV-DDB依赖性损伤识别途径比XPC直接识别占主导地位，并且它们还表明在XPA组装之前NER中存在限速调节步骤。
