Mocquet Vincent, Lainé Jean Philippe, Riedl Thilo, Yajin Zhou, Lee Marietta Y, Egly Jean Marc
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch Cedex, France.
EMBO J. 2008 Jan 9;27(1):155-67. doi: 10.1038/sj.emboj.7601948. Epub 2007 Dec 13.
To address the biochemical mechanisms underlying the coordination between the various proteins required for nucleotide excision repair (NER), we employed the immobilized template system. Using either wild-type or mutated recombinant proteins, we identified the factors involved in the NER process and showed the sequential comings and goings of these factors to the immobilized damaged DNA. Firstly, we found that PCNA and RF-C arrival requires XPF 5' incision. Moreover, the positioning of RF-C is facilitated by RPA and induces XPF release. Concomitantly, XPG leads to PCNA recruitment and stabilization. Our data strongly suggest that this interaction with XPG protects PCNA and Pol delta from the effect of inhibitors such as p21. XPG and RPA are released as soon as Pol delta is recruited by the RF-C/PCNA complex. Finally, a ligation system composed of FEN1 and Ligase I can be recruited to fully restore the DNA. In addition, using XP or trichothiodystrophy patient-derived cell extracts, we were able to diagnose the biochemical defect that may prove to be important for therapeutic purposes.
为了探究核苷酸切除修复（NER）所需的各种蛋白质之间协调作用的生化机制，我们采用了固定模板系统。利用野生型或突变的重组蛋白，我们确定了参与NER过程的因子，并展示了这些因子与固定化受损DNA的相继结合和解离。首先，我们发现增殖细胞核抗原（PCNA）和复制因子C（RF-C）的到达需要XPF进行5'端切割。此外，复制蛋白A（RPA）促进了RF-C的定位，并诱导XPF释放。与此同时，XPG导致PCNA的募集和稳定。我们的数据强烈表明，与XPG的这种相互作用保护PCNA和δ聚合酶免受诸如p21等抑制剂的影响。一旦δ聚合酶被RF-C/PCNA复合物募集，XPG和RPA就会释放。最后，可以募集由翼状内切酶1（FEN1）和连接酶I组成的连接系统来完全修复DNA。此外，使用着色性干皮病（XP）或毛发硫营养不良患者来源的细胞提取物，我们能够诊断出可能对治疗目的很重要的生化缺陷。
