Replication factor C recruits DNA polymerase delta to sites of nucleotide excision repair but is not required for PCNA recruitment.
Nucleotide excision repair (NER) operates through coordinated assembly of repair factors into pre- and postincision complexes. The postincision step of NER includes gap-filling DNA synthesis and ligation. However, the exact composition of this NER-associated DNA synthesis complex in vivo and the dynamic interactions of the factors involved are not well understood. Using immunofluorescence, chromatin immunoprecipitation, and live-cell protein dynamic studies, we show that replication factor C (RFC) is implicated in postincision NER in mammalian cells. Small interfering RNA-mediated knockdown of RFC impairs upstream removal of UV lesions and abrogates the downstream recruitment of DNA polymerase delta. Unexpectedly, RFC appears dispensable for PCNA recruitment yet is required for the subsequent recruitment of DNA polymerases to PCNA, indicating that RFC is essential to stably load the polymerase clamp to start DNA repair synthesis at 3' termini. The kinetic studies are consistent with a model in which RFC exchanges dynamically at sites of repair. However, its persistent localization at stalled NER complexes suggests that RFC remains targeted to the repair complex even after loading of PCNA. We speculate that RFC associates with the downstream 5' phosphate after loading; such interaction would prevent possible signaling events initiated by the RFC-like Rad17 and may assist in unloading of PCNA.
Overmeer
René M
RM
Department of Toxicogenetics, Leiden University Medical Center, Leiden, Netherlands.
Gourdin
Audrey M
AM
Giglia-Mari
Ambra
A
Kool
Hanneke
H
Houtsmuller
Adriaan B
AB
Siegal
Gregg
G
Fousteri
Maria I
MI
Mullenders
Leon H F
LH
Vermeulen
Wim
W
D001665:Binding Sites; D002460:Cell Line; D003561:Cytarabine / Q000494:pharmacology; D004249:DNA Damage; D004258:DNA Polymerase III / Q000378:metabolism*; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication; D036681:Fluorescence Recovery After Photobleaching; D055785:Gene Knockdown Techniques; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D008954:Models, Biological; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D051818:Replication Protein C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D019384:Nucleic Acid Synthesis Inhibitors; D018809:Proliferating Cell Nuclear Antigen; C081632:RFC1 protein, human; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D003561:Cytarabine; D049452:Green Fluorescent Proteins; D004258:DNA Polymerase III; D051818:Replication Protein C; D006918:Hydroxyurea