title: An improved method for the detection of nucleotide excision repair factors at local UV DNA damage sites.
abstract: Among different DNA repair processes that cells use to face with DNA damage, nucleotide excision repair (NER) is particularly important for the removal of a high variety of lesions, including those generated by some antitumor drugs. A number of factors participating in NER, such as the TFIIH complex and the endonuclease XPG are also involved in basal processes, e.g. transcription. For this reason, localization of these factors at DNA damage sites may be difficult. Here we have applied a mild digestion of chromatin with DNase I to improve the in situ extraction necessary to detect chromatin-bound proteins by immunofluorescence. We have compared this method with different extraction protocols and investigated its application on different cell types, and with different antibodies. Our results show that a short DNase I treatment before the immunoreaction, enhances the fluorescence signal of NER proteins, such as XPG, DDB2 and XPC. In addition, our findings indicate that the antibody choice is a critical factor for accurate localization of DNA repair proteins at DNA damage sites. In conclusion, a mild DNA digestion with DNase I improves the immunofluorescence detection of the recruitment of NER factors at local DNA damage sites by enhancing accessibility to the antibodies, independently of the cell type.
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso 207, 27100 Pavia, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Dutto
authors.affiliation: Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università di Pavia, Via Ferrata 9, 27100 Pavia, Italy.
authors.forename: Ornella
authors.initials: O
authors.lastname: Cazzalini
authors.affiliation: Dipartimento di Medicina Molecolare, Unità di Immunologia e Patologia generale, Università di Pavia, Via Ferrata 9, 27100 Pavia, Italy.
authors.forename: Lucia Anna
authors.initials: LA
authors.lastname: Stivala
authors.affiliation: Istituto di Genetica Molecolare del CNR, Via Abbiategrasso 207, 27100 Pavia, Italy. Electronic address: prosperi@igm.cnr.it.
authors.forename: Ennio
authors.initials: E
authors.lastname: Prosperi
mesh_terms: D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000032:analysis* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D004247:DNA; D003850:Deoxyribonuclease I; D045643:DNA Repair Enzymes
keywords: DDB2; Local UV damage; Nucleotide excision repair; PCNA; XPC; XPG