Proc. Natl. Acad. Sci. USA Vol. 90, pp. 6335-6339, July 1993 Medical Sciences DNA excision-repair defect of xeroderma pigmentosum prevents removal of a class of oxygen free radical-induced base lesions (ionizing radiation/endogenous DNA damage/purine dimers/neurological degeneration) MASAHIKO S. SATOH, CHRISTOPHER J. JONES, RICHARD D. WOOD, AND TOMAS LINDAHL Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, United Kingdom Communicated by Philip C. Hanawalt, April 9, 1993 (receivedfor review January 4, 1993) ABSTRACT Plasmid DNA was rirradiated or treated that is initiated by DNA glycosylases present in normal with H202 in the presence of Cu2+ to generate oxygen free amounts in XP cells. We have used an assay measuring DNA radical-induced lesions. Open circular DNA molecules were repair synthesis in damaged DNA added to human cell removed by ethidium bromide/CsCI density gradient centrif- extracts (3) to investigate this problem. It is shown that ugation. The closed circular DNA fraction was treated with the oxygen free radicals induce the formation of a major type of Escherichia coli reagent enzymes endonuclease IH (Nth pro- DNA lesion that cannot be repaired by XP cell extracts. tein) and Fpg protein. This treatment converted DNA mole- cules containing the major base lesions pyrimidine hydrates EXPERIMENTAL PROCEDURES and 8-hydroxyguanine to a nicked form. Remaining closed Cells. Human lymphoblastoid cell lines of normal and XP circular DNA containing other oxygen radical-induced base origin were obtained from the NIGMS Human Genetic Mu- lesions was used as a substrate for nudeotide excision-repair in tant Cell Repository (Camden, NJ). The lymphoblastoid a cell-free system. Extracts from normal human cells, but not XP-G cell line XPG 83 was established in our laboratory. Cell extracts from xeroderma pigmentosum cells, catalyzed repair extracts were made as described (3). synthesis in this DNA. The repair defect in the latter extracts Reagent Enzymes and DNA Substrates. The following pro- could be specifically corrected by in vitro complementation. teins were purified from Escherichia coli overproducer The data suggest that accumulation of endogenous oxidative strains as described: E. coli endonuclease III (Nth protein) damage in cellular DNA from xeroderma pigmentosum pa- (4), E. coli Fpg protein (5), and human XP group A comple- tients contributes to the increased frequency ofinternal cancers menting (XPAC) protein (ref. 6; C.J.J., unpublished data). T4 and the neural degeneration occurring in serious cases of the pyrimidine dimer DNA glycosylase (endonuclease V) was a syndrome. gift from R. S. Lloyd (University of Texas). Plasmid DNA (3.4 mg ml-1 of pBluescript KS+; Stratagene) was y-oirradi- UV light and ionizing radiation induce the formation of ated at 0°C either in 10 mM Tris HCl, pH 8.0/1 mM EDTA different and diverse types of DNA lesions. After exposure (50 or 100 Gy of6OCo t-rays) or in 10 mM sodium phosphate, of DNA to UV light, pyrimidine dimers are the main prod- pH 7.0 (15 Gy of 6OCo trays). Alternatively, plasmid DNA ucts, whereas oxygen free radicals produced by ionizing (0.4 mg-ml-) was treated with 3.2 mM H202/50 ,uM CuS04 for 2 min at 37°C as described by Carmichael et al. (7). In the radiation generate single- and double-strand breaks as well as latter case, the reaction was terminated by addition of a variety of ring-saturated, ring-fragmented, and oxidized catalase (700 units.ml-1; 15 min; 25°C). Covalently closed and base derivatives. Different DNA repair processes are used by open circular forms of DNA were separated by ethidium cells to correct the damage: pyrimidine dimers are removed bromide/CsCl density gradient centrifugation. Incubations as part of oligonucleotides in a nucleotide excision-repair of -t-irradiated or H202/Cu2+-treated closed circular DNA pathway initiated by a multisubunit incision nuclease, while (100 jig-ml-l) with E. coli endonuclease III (1.3 lAg.ml-) were several oxidized bases are liberated in free form by DNA in 40 mM Hepes-KOH, pH 8.0/100 mM KCl/0.5 mM dithio- glycosylases in a base excision-repair process. In cells from threitol/0.2 mg-ml-' of bovine serum albumin for 30 min at patients with the human inherited disease xeroderma pig- 37°C, and incubations of DNA (400 pg.ml-1) with E. coli Fpg mentosum (XP), incision at pyrimidine dimers is defective. protein (2 g'ml-1) were in 70 mM Hepes-KOH, pH 7.8/50 As a consequence, the main symptoms of XP involve skin mM KCl/1 mM EDTA/1 mM dithiothreitol for 1 h at 37°C. and eye lesions occurring after exposure to sunlight, includ- Covalently closed circular DNA remaining after these treat- ing multiple skin cancers. However, other symptoms of XP ments was purified and used as substrate in repair assays. are not readily explained as being due to UV-induced DNA DNA Repair Assays. Rejoining of -irradiated open circular damage. In severe cases, sometimes termed de Sanctis- DNA to a covalently closed form by human cell extracts was Cacchione syndrome, patients exhibit progressive neurolog- measured as described (8). ATP-dependent nucleotide exci- ical deterioration with massive loss of neurons and a 10- to sion-repair by cell extracts and in vitro complementation 20-fold increase in frequency of several types of internal were measured by following DNA repair synthesis as de- cancers (1). The origins of these symptoms have been un- scribed (3, 6). Incubation of H202/Cu2+-treated DNA (100 clear, but they could arise because of accumulation of DNA pg-ml-1) with T4 pyrimidine dimer DNA glycosylase (3 damage generated by exposure to environmental chemical pgml-l) was in 0.1 M NaCl/10 mM Tris HCl, pH 8.0/1 mM mutagens or reactive cellular metabolites (2). In the latter EDTA for 30 min at 37°C. UV-irradiated plasmid DNA (450 regard, DNA is known to undergo endogenous damage due J/m2) was nicked (>98%) under these conditions. to hydrolysis, reaction with active oxygen, and nonenzy- matic methylation. However, the main known lesions intro- RESULTS duced in this fashion are corrected by base excision-repair Rejoining of DNA Single-Strand Breaks. Plasmids were in Tris this converted 20% of the The publication costs ofthis article were defrayed in part by page charge y-irradiated (50 Gy buffer); payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: XP, xeroderma pigmentosum. 6335 Downloaded by guest on September 24, 2021 6336 Medical Sciences: Satoh et al. Proc. Natl. Acad Sci. USA 90 (1993) molecules to an open circular form. The open DNA circles were isolated and incubated with human cell extracts for rejoining into covalently closed circular DNA. The joining reaction was strongly stimulated by NAD+, which promotes the automodification and release of poly(ADP-ribose) poly- CCC DNA merase molecules that bind tightly to strand breaks and | rays inhibit repair in the absence of the cofactor (8). As noted previously (9), only part of the open circular plasmid popu- Pyrimidine glycols lation was rejoined effectively due to end group heterogene- ity. There were no detectable differences in the rates or extents of strand-break rejoining between extracts from ^ f r 8-hydroxyguanine normal cells orXP cells (Fig. 1). Similarresults were obtained and formamidopyrimidines with plasmids treated with H202 in the presence ofCu2+ (data not shown). Repair of DNA Base Lesions. Covalently closed circular 40- DNA exposed to -y-irradiation (100 Gy in Tris buffer, since Other base lesions OC DNA CCC DNA Tris can scavenge hydroxyl radicals, or 15 Gy in phosphate (40%) (60%) buffer) or H202/Cu2+ treatment was recovered, as outlined in Fig. 2, and used as substrate in DNA repair assays with /-A Nth protein human cell extracts (3, 9). At low protein concentrations, there was no discernible difference in repair synthesis be- OC DNA (40%) tween normal and XP cell extracts, whereas XP extracts were consistently slightly less effective at high protein concentra- tions (Fig. 3 A and B; see also Fig. 3 C and D). In agreement with previous results (9), the presence of NAD+ provided a 2- to 3-fold stimulation of DNA repair synthesis, but the CCC DNA (60%) relative difference between normal and XP cells remained. Moreover, a transient appearance of nicked circular DNA /- I Fpg protein was observed (40% conversion to an open circular form after a 10-min incubation at 30°C) and is characteristic of base OC DNA (30%) excision-repair (9). An advantage of the cell-free DNA repair assay is that the plasmid substrate can be altered by stepwise removal of known lesions. Here, we treated the damaged plasmids with CCC DNA (70%) two reagent enzymes, E. coli endonuclease III (pyrimidine hydrate DNA glycosylase; Nth protein) and formamidopy- FIG. 2. Procedure for sequential removal of lesions from DNA rimidine DNA glycosylase (8-hydroxyguanine DNA glyco- exposed to ionizing radiation or H202/Cu2+ treatment. Data for sylase; Fpg protein, MutM protein). These enzymes are percentage conversion of covalently closed circular (CCC) to open universally distributed DNA glycosylases with associated circular (OC) DNA by reagent enzymes were those observed after breaks in treatment of plasmids in Tris buffer with 100 Gy of t.rays; similar abasic lyase activities and generate single-strand results were obtained after exposure ofplasmids in phosphate buffer plasmids containing pyrimidine derivatives lacking a 5,6 to 15 Gy of and after H202/Cu2+ treatment of plasmids. double bond (10) or purines with a fragmented or oxidized t-rays imidazole ring (11). These enzyme treatments converted part fraction, representing -25% of the plasmids treated with 100 of the covalently closed circular DNA to an open circular Gy of y-radiation in Tris buffer or 15 Gy in phosphate buffer, form, and the amounts and relative proportions of Nth and or 30%o of the H202/Cu2+-treated DNA (Fig.