Nucleotide excision repair activity on DNA damage induced by photoactivated methylene blue

• Published in: Free radical biology & medicine Vol. 61; pp. 343 - 356
• Main Authors: Berra, Carolina Maria, de Oliveira, Carla Santos, Garcia, Camila Carrião Machado, Rocha, Clarissa Ribeiro Reily, Lerner, Letícia Koch, Lima, Leonardo Carmo de Andrade, Baptista, Maurício da Silva, Menck, Carlos Frederico Martins
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2013
• Subjects: Cells, Cultured; DNA Damage; DNA Repair; Free radicals; Histones - analysis; Humans; Methylene Blue - pharmacology; Methylene blue photo-excitation; Nucleotide excision repair; Oxidized DNA damage; Phosphorylation; Ultraviolet Rays; Xeroderma Pigmentosum - genetics; XP-A; XP-C; Methylene blue photo-excitation; Nucleotide excision repair; Free radicals; Oxidized DNA damage; XP-A; XP-C
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/j.freeradbiomed.2013.03.026

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 1O2 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. [Display omitted] •NER-deficient cells are sensitive to photoactivated MB and show G2/M accumulation.•NER-deficient cells treated with MB show higher frequency of oxidized DNA lesions.•NER-deficient cells also present more γ-H2AX-stained nuclei after photoactivated MB.•The repair kinetics of oxidized DNA lesions was similar among all cells analyzed.•XPA and XPC proteins have specific roles in protection against ROS-induced DNA damage.