title: Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue.
abstract: The nucleotide excision repair (NER) mechanism is well known to be involved in the removal of UV-induced lesions. Nevertheless, the involvement of this pathway in the repair of lesions generated after DNA oxidation remains controversial. The effects of visible-light-excited methylene blue (MB), known to generate reactive oxygen species (ROS), were examined directly in xeroderma pigmentosum (XP)-A and XP-C NER-deficient human fibroblasts. Initially, MB was confirmed as being incorporated in similar amounts by the cells and that its photoexcitation induces the generation of (1)O2 within cells. The analysis of cell survival indicated that NER-deficient cells were hypersensitive to photoactivated MB. This sensitivity was confirmed with cells silenced for the XPC gene and by host-cell reactivation (HCR) of plasmid exposed to the photosensitizing effects of photoexcited MB. The sensitivity detected by HCR was restored in complemented cells, confirming the participation of XPA and XPC proteins in the repair of DNA lesions induced by photosensitized MB. Furthermore, DNA damage (single- and double-strand breaks and alkali-sensitive sites) was observed in the nuclei of treated cells by alkaline comet assay, with higher frequency of lesions in NER-deficient than in NER-proficient cells. Likewise, NER-deficient cells also presented more γ-H2AX-stained nuclei and G2/M arrest after photoactivated MB treatment, probably as a consequence of DNA damage response. Notwithstanding, the kinetics of both alkali- and FPG-sensitive sites repair were similar among cells, thereby demonstrating not only that MB photoexcitation generates nuclear DNA damage, but also that the removal of these lesions is NER-independent. Therefore, this work provides further evidence that XPA and XPC proteins have specific roles in cell protection and repair/tolerance of ROS-induced DNA damage. Moreover, as XPC-deficient patients do not present neurodegeneration, premature aging, or developmental clinical symptoms, the results indicate that defects in the repair/tolerance of oxidatively generated DNA lesions are not sufficient to explain these severe clinical features of certain XP patients.
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Carolina Maria
authors.initials: CM
authors.lastname: Berra
authors.affiliation: Center of Health and Biological Sciences, University of Mato Grosso do Sul, Campo Grande, MS, Brazil.
authors.forename: Carla Santos
authors.initials: CS
authors.lastname: de Oliveira
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Camila Carrião Machado
authors.initials: CC
authors.lastname: Garcia
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Clarissa Ribeiro Reily
authors.initials: CR
authors.lastname: Rocha
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Letícia Koch
authors.initials: LK
authors.lastname: Lerner
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Leonardo Carmo de Andrade
authors.initials: LC
authors.lastname: Lima
authors.affiliation: Department of Biochemistry, Institute of Chemistry, University of São Paulo, São Paulo, SP 05508-900, Brazil.
authors.forename: Maurício da Silva
authors.initials: Mda S
authors.lastname: Baptista
authors.affiliation: Department of Microbiology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil. Electronic address: cfmmenck@usp.br.
authors.forename: Carlos Frederico Martins
authors.initials: CF
authors.lastname: Menck
mesh_terms: D002478:Cells, Cultured; D004249:DNA Damage; D004260:DNA Repair; D006657:Histones / Q000032:analysis; D006801:Humans; D008751:Methylene Blue / Q000494:pharmacology*; D010766:Phosphorylation; D014466:Ultraviolet Rays; D014983:Xeroderma Pigmentosum / Q000235:genetics
chemical_list: C468783:H2AX protein, human; D006657:Histones; D008751:Methylene Blue
keywords: Free radicals; Methylene blue photo-excitation; Nucleotide excision repair; Oxidized DNA damage; XP-A; XP-C