Sequential recruitment of the repair factors during NER: the role of XPG in initiating the resynthesis step.
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.
Mocquet
Vincent
V
Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch Cedex, France.
Lainé
Jean Philippe
JP
Riedl
Thilo
T
Yajin
Zhou
Z
Lee
Marietta Y
MY
Egly
Jean Marc
JM
D002460:Cell Line; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000502:physiology; D004249:DNA Damage / Q000502:physiology; D004258:DNA Polymerase III / Q000037:antagonists & inhibitors / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D045643:DNA Repair Enzymes / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000502:physiology*; D004720:Endonucleases / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000502:physiology*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology*; D014466:Ultraviolet Rays; D000099250:DNA Excision Repair Protein ERCC-5
D050759:Cyclin-Dependent Kinase Inhibitor p21; D004258:DNA Polymerase III; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C493956:CDKN1A protein, human