title: 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. Furthermore, 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 process in vivo in 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.
authors.affiliation: Department of Dental Radiology and Radiation Research, Faculty of Dentistry, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Miura
authors.forename: S
authors.initials: S
authors.lastname: Nakamura
authors.forename: T
authors.initials: T
authors.lastname: Sasaki
authors.forename: Y
authors.initials: Y
authors.lastname: Takasaki
authors.forename: T
authors.initials: T
authors.lastname: Shiomi
authors.forename: M
authors.initials: M
authors.lastname: Yamaizumi
mesh_terms: D000818:Animals; D000918:Antibody Specificity; D002459:Cell Fusion / Q000502:physiology; D002460:Cell Line / Q000737:chemistry / Q000201:enzymology / Q000528:radiation effects; D004249:DNA Damage / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000737:chemistry / Q000201:enzymology / Q000528:radiation effects; D005455:Fluorescent Antibody Technique; D005816:Genetic Complementation Test; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D010957:Plasmids; D018809:Proliferating Cell Nuclear Antigen / Q000032:analysis* / Q000276:immunology; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors; D014162:Transfection; D014466:Ultraviolet Rays; D000099250:DNA Excision Repair Protein ERCC-5
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D004720:Endonucleases; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D011506:Proteins; D014157:Transcription Factors; D000099250:DNA Excision Repair Protein ERCC-5; C071447:ERCC1 protein, human; C499360:Ercc1 protein, mouse