Roles of XPG and XPF/ERCC1 Endonucleases in UV-Induced Immunostaining of PCNA in Fibroblasts

Abstract

To investigate the relationship between proliferating cell nuclear antigen (PCNA) complex formation and dual incisions in the nucleotide excision repair (NER) process, xeroderma pigmentosum group G (XP-G), XP-F, and XP-G equivalent mouse UV-sensitive mutant ERCC group 5 cells were utilized as a model in this study. These cells are deficient in endonucleases related to 3′ (XP-G and ERCC group 5) or 5′ (XP-F) incision of the DNA lesions in the NER process. PCNA complex formation was detected by an indirect immunofluorescence method after the cells were fixed in methanol. When Sps1 (XP-G) and XL216-7 (ERCC group 5) cells were UV irradiated, neither of them showed PCNA staining. In contrast, SFN4 (a human normal strain) and heterokaryons of Sps1 and XP96TO (XP-A) cells fused by polyethylene glycol treatment showed PCNA staining following UV irradiation. Further- more, XLgfPAneo1 cells, derived from XL216-7 cells transfected with a plasmid containing mouse ERCC5 (xpg) cDNA, also restored staining and UV sensitivity. On the other hand, we observed a very faint PCNA staining in XP2YO (XP-F) cells, expressing no detectable ERCC1 or XPF protein, after UV irradiation. X rays induced PCNA staining in all cell lines with a similar staining pattern, and radiosensitivity was exactly the same between XL216-7 and XLgfPAneo1 cells. These results may have implications for the NER processin vivoin that coordinately occurring dual incisions by XPG and XPF/ERCC1 proteins play an important role in inducing PCNA complex formation, but the step may not be required for PCNA-dependent repair of X-ray-induced DNA damage.