Replication Factor C Recruits DNA Polymerase δ 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

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. A multitude of endogenous and exogenous genotoxic agents induce damage to DNA. When not repaired properly, these DNA lesions can interfere with replication and transcription and thereby induce deleterious events (i.e., cell death, mutations, and genomic instability) that affect the fate of organisms (18). To ensure the maintenance of the DNA helix integrity, a network of defense mechanisms has evolved including accurate and efficient DNA repair processes. One of these processes is the nucleotide excision repair (NER) pathway that removes a wide range of DNA helix-distorting lesions, such as sunlightinduced photodimers, for example, cyclobutane pyrimidine dimers (CPD) and pyrimidine 6-4 pyrimidone photoproducts (6-4PP). Within NER, more than 30 polypeptides act coordinately, starting from the detection and removal of the lesion up to the restoration of the DNA sequence and chromatin structure. The importance of NER is underlined by the severe clinical consequences associated with inherited NER defects, causing UV-hypersensitive autosomal recessive syndromes: the cancer-predisposing xeroderma pigmentosum (XP) and the premature ageing and neurodegenerative disorders Cockayne syndrome (CS) and trichothiodystrophy (TTD) (27). The initial DNA damage recognition step in NER involves two subpathways: transcription-coupled repair (TCR) and global genome repair (GGR). TCR is responsible for the rapid removal of transcription-blocking DNA lesions and is initiated when elongating RNA polymerase II stalls at a DNA lesion on the transcribed strand (16). In GGR, which removes lesions throughout the genome, damage recognition is facilitated by the concerted action of the heterodimeric XP group C (XPC)-HR23B protein complex and by the UV-damaged DNA-binding protein (UV-DDB) complex (10, 33). Subsequently, the 10-subunit TFIIH complex unwinds the DNA around the lesion. This partially unwound structure is stabilized by the single-strand binding protein replication protein A (RPA) and the damage-verifying protein XPA. Collectively, these proteins load and properly orient the structure-specific endonucleases XPF-ERCC1 and XPG that incise 5Ј and 3Ј of the damage, respectively, creating a single-strand gap of approximately 30 nucleotides (nt