Reactive oxygen-mediated damage to a human DNA replication and repair protein

Abstract

Ultraviolet A (UVA) makes up more than 90% of incident terrestrial ultraviolet radiation. Unlike shorter wavelength UVB, which damages DNA directly, UVA is absorbed poorly by DNA and is therefore considered to be less hazardous. Organ transplant patients treated with the immunosuppressant azathioprine frequently develop skin cancer. Their DNA contains 6-thioguanine-a base analogue that generates DNA-damaging singlet oxygen ((1)O(2)) when exposed to UVA. Here, we show that this (1)O(2) damages proliferating cell nuclear antigen (PCNA), the homotrimeric DNA polymerase sliding clamp. It causes covalent oxidative crosslinking between the PCNA subunits through a histidine residue in the intersubunit domain. Crosslinking also occurs after treatment with higher-although still moderate-doses of UVA alone or with chemical oxidants. Chronic accumulation of oxidized proteins is linked to neurodegenerative disorders and ageing. Our findings identify oxidative damage to an important DNA replication and repair protein as a previously unrecognized hazard of acute oxidative stress.

Figure 1

Formation and characterization of a new high-molecular-weight PCNA species. (A) CCF-CEM (left panel) or HCT116 (right panel) cells were grown for 48 h in medium containing 6-TG at the concentrations indicated and irradiated with 3 kJ/m² UVA. Both cell lines are mismatch repair defective and accumulate significant levels of DNA 6-TG (≥0.1% of DNA guanine) without toxic effect; control cells were unirradiated. HCT116 cells grown in the absence of 6-TG were also treated with H₂O₂ (0.03%, 2 min) or 10 J/m² UVC. Chromatin-associated proteins extracted 6 h after irradiation, or immediately after H₂O₂ treatment, were analysed by western blotting with PC10 PCNA antibodies. The position of monoubiquitinated PCNA (Ubi-PCNA) is indicated. Immunoprecipitation experiments followed by western blotting with ubiquitin antibodies confirmed the presence of ubiquitin. The 90 kDa PCNA complex (PCNA^*) is also shown. This complex migrated slightly more slowly than a nonspecific band that is more prominent in HCT116 than in CCF-CEM extracts. (B) Kinetics of PCNA^* formation. HCT116 cells grown for 48 h in 1 μM 6-TG were irradiated with 1 kJ/m² UVA at a high dose rate (0.5 kJ/m²/s). Extracts prepared at the time points indicated were analysed by western blotting with PC10. (C) PCNA^* pI. Chromatin-associated proteins from CCF-CEM cells grown in the presence or absence of 6-TG (1 μM, 48 h), as indicated, and UVA irradiated (10 kJ/m²) were analysed by two-dimensional IEF/PAGE analysis followed by western blotting with PC10. (D) Size-exclusion chromatography. Chromatin-associated proteins extracted from CCF-CEM cells treated with 6-TG (1 μM, 48 h) and UVA (50 kJ/m²) were separated by chromatography on AcA44 precalibrated with alcohol dehydrogenase (150 kDa), BSA (66 kDa) and carbonic anhydrase (30 kDa). Fractions containing PCNA were identified by dot blot and analysed further by western blotting with PC10. 6-TG, 6-thioguanine; Control, unirradiated; IEF/PAGE, isoelectric focusing-polyacrylamide gel electrophoresis; PCNA, proliferating cell nuclear antigen; UVA, ultraviolet A.

Figure 2

Formation of a new high-molecular-weight PCNA species at active replication forks. (A) The 90 kDa PCNA complex (PCNA^*) in non-S- compared with S-phase cells. Synchronized HCT116 cells were 6-TG labelled (60 min, 10 μM) 2 h after release from a double thymidine block. At this time ≥90% of cells were in S phase, as determined by FACS of propidium iodide (PI)-stained cells (upper panel). Cell-cycle distributions were assigned using Watson Pragmatic. Aliquots of 6-TG-treated cells were UVA irradiated (10 kJ/m²) either 9 h (when 85% of cells were in G₁+G₂ phase) or at 16 h (>80% S phase) after 6-TG treatment. Cell extracts were analysed by western blots probed with PC10 (lower panel); the positions of PCNA and PCNA^* are indicated. Left: G₁+G₂ cells; right: S-phase cells. (B) 6-TG pulse–chase. Exponentially growing CCF-CEM cells were pulse labelled with 6-TG (10 μM, 15 min), followed by a return to normal growth medium without 6-TG. At the time points indicated, aliquots of cells were irradiated with UVA (10 kJ/m²). Immediately after irradiation, one sample of each aliquot was used to monitor DNA replication by [³H]thymidine incorporation, which is expressed as a percentage of that in unirradiated cells. The remaining cells were extracted and proteins analysed by western blotting with PC10. 6-TG, 6-thioguanine; A, asynchronous untreated cells; D, double thymidine block only; FACS, fluorescence-activated cell sorting; PCNA, proliferating cell nuclear antigen; T, 6-TG only; T+U, 6-TG+UVA; U, UVA only; UVA, ultraviolet A.

Figure 3

Photochemical formation of a new high-molecular-weight PCNA species requires a conserved histidine residue. (A) Crosslinking by UVA alone. Left panel: asynchronous A2780 cells grown in the presence or absence of 6-TG (0.2 μM, 48 h) were UVA irradiated as indicated; cell extracts were analysed by western blotting with PC10. Right panel: Raji cells synchronized by DMSO (2%, 48 h) were UVA (kJ/m²) or UVC (J/m²) irradiated as indicated in either G₁ or S phase. PCNA was analysed by western blotting. G₁ irradiated 7 h after release (>90% G₀/G₁ by PI staining and FACS analysis); S irradiated 20 h after release (>90% S phase). (B) Intersubunit region of human PCNA with alignment of conserved residues with reactive side chains. Lys 77 and Lys 110, and Arg 146 and Arg 149 are highly conserved; His 153 is replaced by glutamine in the insect Spodoptera frugiperda and in Xenopus laevis. Other potentially reactive intersubunit residues—Cys 81, Cys 148 and Met 116—are fully conserved. (C) Chemical and photochemical PCNA crosslinking. Human HCT116 or insect SF9 cells were treated for 15 min with formaldehyde or UVA irradiated. X. laevis egg extracts (Kubota & Takisawa, 1993) were induced to replicate by the addition of sperm nuclei. After 5 min, at which time point there was minimal replication (Replication−) as indicated by nucleus formation (Hoechst staining) and fluorescent dAMP from Cy3-ATP incorporation (dATP), or after 45 min, during the period of maximal DNA replication with significant dAMP incorporation into well-defined nuclei (Replication+), extracts were UVA irradiated. An aliquot of the same replication+extract was treated with formaldehyde. HCT116 cells treated with 6-TG (0.1 μM, 24 h) and irradiated with UVA (10 kJ/m²) were included as a control, C. Cell and egg extracts were analysed by western blotting (6% SDS–PAGE) using the PC10 antibody as probe. 6-TG, 6-thioguanine; DMSO, dimethyl sulphoxide; FACS, fluorescence-activated cell sorting; PCNA, proliferating cell nuclear antigen; PI, propidium iodide; UVA, ultraviolet A.