Li R Y, Calsou P, Jones C J, Salles B
Société Française de Recherches et d'Investissements (SFRI), Berganton, Saint Jean d'Illac, 33127, France.
J Mol Biol. 1998 Aug 14;281(2):211-8. doi: 10.1006/jmbi.1998.1949.
We have studied the interactions between DNA damage and human proteins involved in the early steps of nucleotide excision repair (NER) reaction under in vitro conditions with human protein extracts. By using a new assay, we have detected a long-lived DNA/protein complex involving XPA and TFIIH in the course of the NER process. The formation of this complex is exclusively limited to DNA lesions that are substrates of the human excinuclease. We show that, while XPA binding to damaged DNA is ATP-independent, stable association of TFIIH with DNA lesions is promoted by ATP hydrolysis and is dependent on the integrity of XPA and XPC proteins in the cell extract. In addition, XPC is necessary to promote a stable binding of XPA to UV-irradiated DNA. Finally, the co-binding of XPA and TFIIH to DNA damage is correlated to a dose-dependent titration of TFIIH and not XPA from the free protein fraction.
我们利用人蛋白质提取物，在体外条件下研究了DNA损伤与参与核苷酸切除修复（NER）反应早期步骤的人类蛋白质之间的相互作用。通过使用一种新的检测方法，我们在NER过程中检测到了一种涉及XPA和TFIIH的长寿命DNA/蛋白质复合物。这种复合物的形成仅局限于作为人类核酸内切酶底物的DNA损伤。我们发现，虽然XPA与受损DNA的结合不依赖ATP，但TFIIH与DNA损伤的稳定结合是由ATP水解促进的，并且依赖于细胞提取物中XPA和XPC蛋白质的完整性。此外，XPC对于促进XPA与紫外线照射的DNA的稳定结合是必需的。最后，XPA和TFIIH与DNA损伤的共结合与TFIIH而非XPA从游离蛋白质组分中的剂量依赖性滴定相关。
