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Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress
Article first published online: 17 JAN 2013
© 2012 Wiley Periodicals, Inc.
Volume 34, Issue 3, pages 481–489, March 2013
How to Cite
Soltys, D. T., Rocha, C. R. R., Lerner, L. K., de Souza, T. A., Munford, V., Cabral, F., Nardo, T., Stefanini, M., Sarasin, A., Cabral-Neto, J. B. and Menck, C. F. M. (2013), Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress. Hum. Mutat., 34: 481–489. doi: 10.1002/humu.22259
Communicated by Riccardo Fodde
- Issue published online: 18 FEB 2013
- Article first published online: 17 JAN 2013
- Accepted manuscript online: 15 DEC 2012 10:10PM EST
- Manuscript Accepted: 30 NOV 2012
- Manuscript Received: 29 JUL 2012
- Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil)
- Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília, DF, Brazil)
- Associazione Italiana per la Ricerca sul Cancro (Milan, Italy)
Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.
Figure S1. Increased levels of sub-G1 population in XP-G fibroblasts after UV irradiation but not oxidative stress. A: Representative histograms of UV-induced apoptosis in XP01RJ and XPHF01RJ cells 72 hours after 14.0 J/m2 UV irradiation. B: Representative histograms of apoptosis in XP01RJ (left panel) and XPCS1LV cells (right panel) treated with 8 μM of photosensitised MB. The X-axis indicates the amount of DNA (PI fluorescence), and the Y-axis indicates the number of cells. The numbers are the percentages of cells in the sub-G1 population.
Figure S2. Transcriptional and translational levels of XPG are normal in XP01RJ fibroblasts. A: XPG mRNA expression was evaluated by quantitative RT-PCR in total RNA samples obtained from XP01RJ (XP-G), XPHF01RJ (mother of the patient XP01RJ) and NHF cells (wild type), either unirradiated (dark grey columns) or 5 hours after UV irradiation in equitoxic doses (bright grey columns). The NHF non-irradiated sample was used as a reference, and β-actin was the loading control. The columns represent the mean of two independent experiments performed in triplicate. Error bars indicate SD. B: XPG protein levels were assessed by western of UV irradiated or control XP01RJ, NHF, and XP05SP (XP-V) fibroblasts. Tubulin was used as the loading control.
Figure S3. Absence of exon skipping in the XPG mRNA of XP01RJ and XP02RJ fibroblasts. A: Schematic representation of amplicons designed to check possible exon skipping in the XPG mRNA. The numbers inside the central rectangle represents the number of the exon of XPG gene. Red and green arrows represent the primers that were used to generate the respective amplicon. B: Agarose electrophoresis for the amplicons generated with RT-PCR reactions designed in (A) and the mRNA samples: 1 - NHF; 2 - XPHF01RJ; 3 - XP02RJ; 4 - XP01RJ; 5 - negative control. DNA samples from these fibroblasts were used as a control. No difference was observed among the different cell lines, indicating that there is no exon skipping in these fibroblasts.
Figure S4. Two novel XPG mutations cause the XP phenotype of the Brazilian patients. Mutations c.83C>A (A) and c.2904G>C (B) in the XPG gene of the Brazilian XP-G patients (XP01RJ and XP02RJ) and their mother (XPHF01RJ). Shown here are representative chromatograms of the sequencing of both the cDNA and genomic DNA obtained from these cell lines. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.
Figure S5. Ramachandran plots from RAMPAGE of selected XPG (A) and XPG-Trp968Cys (B) models.
Table S1. Primers used in this work with main characteristics
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