Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells Melisa Guven, Reto Brem, Peter Macpherson, Matthew Peacock, Peter.

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Oxidative Damage to RPA Limits the Nucleotide Excision Repair Capacity of Human Cells Melisa Guven, Reto Brem, Peter Macpherson, Matthew Peacock, Peter Karran Journal of Investigative Dermatology Volume 135, Issue 11, Pages 2834-2841 (November 2015) DOI: 10.1038/jid.2015.255 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Replication protein A (RPA) overexpression, nucleotide excision repair (NER), and sensitivity. (a) Western blotting for RPA14, RPA32, and RPA70. (b) 6:4 Py:Py ELISA of 6-TG-treated U2OS and U2OS-RPA21 cells irradiated with UVA (20kJm-2)+10Jm-2 UVC. (∆ ▲) 10Jm-2 UVC alone. Mean values from ≥3 experiments. (c) Viability of UVA-irradiated cells with Lo (~0.06%) or Hi (~0.14%) DNA 6-thioguanine (6-TG). Mean values from two experiments. (d) 6:4 Py:Py ELISA of cells treated with ciprofloxacin (CIP; 1hour; 0.5mm)+UVA (20kJm-2)+UVC (10Jm-2). Mean values from ≥3 experiments. (e) As in d, cells treated with 1mm ofloxacin, UVA+UVC. (f) Ciprofloxacin/UVA toxicity. Viability of cells treated for 1hour with ciprofloxacin and UVA (20kJm-2). Mean values from ≥3 experiments. (g) Ofloxacin/UVA toxicity. As in f, cells were treated with ofloxacin and UVA. Journal of Investigative Dermatology 2015 135, 2834-2841DOI: (10.1038/jid.2015.255) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Replication protein A (RPA) and DDB2 chromatinization following treatment. (a) 6-TG/UVA. HeLa, U2OS, or U2OS-RPA21 cells grown for 24hours in 1, 4, or 10μm 6-TG, respectively, to ensure equivalent DNA substitution were UVA irradiated (20kJm-2). Whole-cell extracts were analyzed by western blotting for RPA70. (b) Ciprofloxacin/UVA and Ofloxacin/UVA. U2OS or U2OS-RPA21 cells were treated with 0.5mm ciprofloxacin or ofloxacin as indicated and irradiated with UVA (20kJm-2). Whole-cell extracts were analyzed as in a. (c) DDB2. U2OS cells treated with 6-thioguanine (6-TG) for 24hours or fluoroquinolone for 1hour at the concentrations shown were UVA irradiated as indicated. Whole-cell extracts prepared immediately after irradiation were analyzed by western blotting for DDB2 or DDB1/ β-actin as loading controls. Journal of Investigative Dermatology 2015 135, 2834-2841DOI: (10.1038/jid.2015.255) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Formation of RPA32 complexes. (a, Left panel). RPA32 western blots of extracts from U2OS cells either untreated or treated with hydroxyurea (HU; 3mm, 3hours), ionizing radiation (IR; 20Gy), UVC (100Jm-2), or 6-thioguanine (6-TG)/UVA (2 or 4μm 6-TG, 24h/20kJm-2). (Right panel) RPA32 western blots of U2OS-RPA21 cells treated identically, except that 6-TG concentrations were 4 or 8μm 6-TG to ensure similar levels of DNA 6-TG. Allopurinol (1mm) was included during 6-TG treatment as indicated. RPA32 complexes are indicated (* and **). (b) RPA32 western blots of extracts from U2OS and U2OS-RPA21 cells treated for 1hour with ciprofloxacin/UVA (20kJm-2) as indicated. MW markers and RPA32 complexes (*, **, and ***) are indicated. (c) As in b. Cells treated with ofloxacin/UVA (20kJm-2). Journal of Investigative Dermatology 2015 135, 2834-2841DOI: (10.1038/jid.2015.255) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Identity of replication protein A (RPA) oxidation products. (a) Immunoprecipitation. Anti-RPA32 immunoprecipitates from U2OS-RPA21 cells treated with ciprofloxacin (0.5mm, 1hour) and UVA (20kJm-2) were analyzed by western blotting for RPA32 and RPA70. The two lower panels represent longer exposure of the boxed areas 1 and 2. Positions of molecular weight markers are indicated. (b) RPA32 cysteine sulfenate. U2OS-RPA21 cells that had been treated with ciprofloxacin (250μm, 1hour) and UVA (20kJm-2) were lysed in the presence of the BP-1 biotinylated sulfenate-reactive probe. Derivatized proteins were recovered from streptavidin-linked beads (Eluate) and analyzed by western blotting for RPA32. Samples before streptavidin enrichment (Input) were also analyzed. (c) Extracts from U2OS-RPA21 cells were treated with ofloxacin (OFL 500μm, 1hour) and UVA (20kJm-2) and analyzed as in b. Journal of Investigative Dermatology 2015 135, 2834-2841DOI: (10.1038/jid.2015.255) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Nucleotide excision repair (NER) in vitro. (a) NER assays of nuclear extracts from U2OS or U2OS-RPA21 cells treated (1hour) with ciprofloxacin (0.5mm) or ofloxacin (1mm) and irradiated with 20kJm-2 UVA as indicated. Excision products (indicated) were radiolabeled and analyzed by urea gel electrophoresis (upper panels). The same nuclear extracts were analyzed by western blotting (lower panel). The position of the treatment-related RPA32 complex is indicated as *. (b) Excision products were quantitated by GelDoc, and NER efficiency by extracts from drug/UVA-treated cells is expressed relative to that of extracts from cells treated with drug alone. Means of three to five assays of independently prepared extracts. Journal of Investigative Dermatology 2015 135, 2834-2841DOI: (10.1038/jid.2015.255) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions