Mitochondrial Repair of 8-Oxoguanine Is Deficient in Cockayne Syndrome

Oncogene (2002) 21, 8675 – 8682 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc ORIGINAL PAPERS Mitochondrial repair of 8-oxoguanine is deﬁcient in Cockayne syndrome group B

Tinna Stevnsner1, Simon Nyaga2, Nadja C de Souza-Pinto2, Gijsbertus TJ van der Horst3, Theo GMF Gorgels3,4, Barbara A Hogue2, Tina Thorslund1 and Vilhelm A Bohr*,2

1Danish Center for Molecular Gerontology, Department of Molecular Biology, University of Aarhus, DK-8000 Aarhus C, Denmark; 2Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, MD 21224, USA; 3Medical Genetics Centre Department of Cell Biology and Genetics, Center for Biomedical Genetics, Erasmus University Rotterdam, 3000 DR, Rotterdam, The Netherlands

Reactive oxygen species, which are prevalent in premature aging (Friedberg, 1996; Nance and Berry, mitochondria, cause oxidative DNA damage including 1992). Cells from CS patients are more sensitive than the mutagenic DNA lesion 7,8-dihydroxyguanine (8- wild type cells to DNA damage induced by ultraviolet oxoG). Oxidative damage to mitochondrial DNA has (UV) light (Wade and Chu, 1979), 4-Nitroquinoline-1- been implicated as a causative factor in a wide variety of oxide (4NQO) (Wade and Chu, 1979), N-acetoxy-2- degenerative diseases, and in cancer and aging. 8-oxoG acetylaminofluorene (NA-AAF) (van Oosterwijk et al., is repaired efficiently in mammalian mitochondrial DNA 1996), ionizing radiation, and hydrogen peroxide by enzymes in the base excision repair pathway, (Cooper et al., 1997; Leadon and Cooper, 1993). CS including the 8-oxoguanine glycosylase (OGG1), which cells are deficient in transcription coupled repair (TCR) incizes the lesion in the first step of repair. Cockayne of lesions caused by UV irradiation (Evans and Bohr, syndrome (CS) is a segmental premature aging syndrome 1994; Venema et al., 1990) or oxidative stress (Cooper in humans that has two complementation groups, CSA et al., 1997; Le Page et al., 2000). Furthermore, RNA and CSB. Previous studies showed that CSB-deficient synthesis recovers more slowly in UV-treated CS cells cells have reduced capacity to repair 8-oxoG. This study than in wild type cells (Mayne and Lehmann, 1982). examines the role of the CSB gene in regulating repair CS cells also have a stronger apoptotic response to UV of 8-oxoG in mitochondrial DNA in human and mouse exposure than wild type cells (Ljungman and Zhang, cells. 8-oxoG repair was measured in liver cells from 1996). CSB deficient mice and in human CS-B cells carrying There are two CS complementation groups, CS-A expression vectors for wild type or mutant forms of the and CS-B (Henning et al., 1995; Lehmann, 1982; human CSB gene. For the first time we report that CSB Tanaka et al., 1981; Troelstra et al., 1992). The CSB stimulates repair of 8-oxoG in mammalian mitochondrial gene encodes a 168 kD protein (Troelstra et al., 1992) DNA. Furthermore, evidence is presented to support the that has homology to the SWI/SNF family of proteins. hypothesis that wild type CSB regulates expression of CSB has an acidic region, a glycine rich region, two OGG1. putative nuclear localization signal sequences, and an Oncogene (2002) 21, 8675 – 8682. doi:10.1038/sj.onc. ATPase domain. The ATPase domain includes seven 1205994 conserved ATPase motifs found in all SWI/SNF family proteins. The CSB ATPase domain supports DNA Keywords: base excision repair; oxidative damage; stimulated ATPase activity, but does not support DNA mitochondria; Cockayne syndrome helicase activity, as measured by a conventional strand displacement assay (Selby and Sancar, 1997b; Tantin et al., 1997). Previous studies show that the CSB ATPase Introduction domain plays a role in regulating the apoptotic response to UV exposure (Balajee et al., 2000) and Cockayne syndrome (CS) is a rare autosomal recessive plays an essential role in TCR of UV-induced DNA disorder associated with sensitivity to sunlight, severe damage as well as in a TCR-independent nucleotide neurological abnormalities, dwarfism, and signs of excision repair (NER) pathway (Brosh et al., 1999). SWI/SNF proteins participate in several biological processes including regulation of transcription, DNA *Correspondence: VA Bohr; Chief Laboratory of Molecular repair and maintenance of chromosome stability (Eisen Gerontology, Box 1, National Institute on Aging, IRP, NIH 5600 et al., 1995). However, the precise biological role(s) and Nathan Shock Drive, Baltimore, MD 21224-6825; molecular mechanism of CSB is not clear. For E-mail: [email protected] example, in vivo and in vitro evidence supports a role 4Current address: Netherlands Ophtalmic Research Institute, KNAW Meibergdreef 47, 1100 BA, Amsterdam, The Netherlands for CSB in the process of transcription (Balajee et al., Received 17 June 2002; revised 19 August 2002; accepted 20 1997; Selby and Sancar, 1997a). CSB also interacts August 2002 directly with core histones and may play a role in ATP- Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8676 dependent chromatin remodeling (Citterio et al., 2000). or repair of 8-oxoG in mouse cells deficient in CSB or Other studies support the idea that CSB plays a direct human cells expressing wild type or an ATPase- or indirect role in the repair of some types of oxidative deficient form of CSB. Evidence is presented to support DNA damage (Cooper et al., 1997; Dianov et al., the conclusion that wild type CSB stimulates repair of 1999). 8-oxoG in mitochondrial DNA. This stimulation does Reactive oxygen species (ROS) are formed in all not require the ATPase activity of CSB and may be living cells as a by-product of normal metabolism mediated by increased expression of OGG1 protein. (endogenous sources) and following exposure to environmental compounds (exogenous sources). Endo- Results genous ROS are largely formed during oxidative phosphorylation in the mitochondria of eukaryotic Incision of 8-oxoG by mitochondrial extracts of human cells and mitochondrial DNA is at particularly high CS-B cells risk of ROS-induced damage. One of the most common oxidative DNA lesions is 7,8-dihydroxygua- In order to assess the role of the CSB gene product in nine (8-oxoG). Mitochondria efficiently repair 8-oxoG mitochondrial DNA repair a complementation study in and other damage to DNA bases (Anson et al., 1998; the human CS-B cell line, CS1AN, was performed. LeDoux et al., 1992; Taffe et al., 1996), but have no CS1AN cells were stably transfected with a construct capacity to repair UV-induced pyrimidine dimers expressing wild type CSB (CS1AN/pc3.1-CSBwt) or (Clayton et al., 1974). CSB with a point mutation that completely inactivates 8-oxoG is repaired primarily by the base excision the CSB ATPase activity (CS1AN/pc3.1-CSBE646Q) repair (BER) pathway (Dianov et al., 1998). In (Figure 1 and our unpublished data). Clones expressing mammalian cells, the 8-oxoG glycosylase, OGG1, CSB protein at the same level as wild type cells were carries out the first step of repair of 8-oxoG: OGG1 chosen for further analysis. As a control CS1AN cells recognizes the damaged base and cleaves the N- were stably transfected with the expression vector glycosylic bond, releasing the damaged base and (CS1AN/pc3.1). Previous studies showed that the forming an apurinic/apyrimidinic (AP) site. The AP CS1AN cells and the CS1AN/pc3.1 cell line are site is rapidly nicked by an AP endonuclease or by the sensitive to UV irradiation while expression of the AP lyase activity associated with OGG1 (Bjoras et al., wild type CSB gene restores normal UV-resistance to 1997; Roldan-Arjona et al., 1997). OGG1 is encoded CS1AN cells (Selzer et al., 2002). However, cells by a nuclear gene, and alternative splicing of pre- expressing the ATPase deficient CSB protein mRNA gives rise to at least two isoforms of OGG1, (CS1AN/pc3.1-CSBE646Q) exhibit an UV sensitivity which are transported to the nucleus and the and apoptotic response similar to CS-B cells (Balajee et mitochondria, respectively. OGG1 knockout mice were al., 2000; Brosh et al., 1999; Selzer et al., 2002). recently generated. In these animals, 8-oxoG is not Mitochondrial extracts were prepared from the removed from the mitochondrial DNA of liver cells (de established cell lines and assayed for ability to incize Souza-Pinto et al., 2001). Other studies suggest that a 28 bp oligonucleotide substrate with a single 8-oxoG repair of 8-oxoG is deficient in extracts of CS-B cells, residue at position 11 (Table 1). In the mitochondrial and that transcription of OGG1 may be decreased in extract of CS1AN/pc3.1 cells, 10+1% of the substrate these cells (Dianov et al., 1999). These results indicate a oligonucleotide was incized at the 8-oxoG base (Figure possible role for CSB in base excision repair of 2a,b). The amount of 8-oxoG incision activity oxidative damage. increased approximately twofold in cells expressing This study explores the hypothesis that CSB plays a wild type CSB (19+2% incision) and also in the cells role in regulating repair of 8-oxoG in mitochondrial expressing the ATPase deficient CSB mutant (18+4%). DNA. This hypothesis is supported by the observation The increased incision activity was statistically signifi- that clinical features of CS are reminiscent of cant (P50.005). Similar results were observed using a syndromes that involve mitochondrial dysfunction. different 8-oxoG-containing oligonucleotide substrate One possible explanation of this observation is that (data not shown). This result suggests that the CSB mitochondrial DNA damage accumulates in CS-B gene product stimulates repair of 8-oxoG in mitochon- cells. These ideas were tested by measuring incision dria in an ATPase-independent manner.

Table 1 Olignucleotide substrates employed in this study 8-oxoG 5’-GAACGACTGTOACTTGACTGCTACTGAT-3’ 3’-CTTGCTGACACTGAACTGACGATGACTA-5’ Uracil 5’-ATATACCGCGGUCGGCCGATCAAGCTTATT-3’ 3’-TATATGGCGCCCGCCGGCTAGTTCGAATAA-5’ hX 5’-AATTGCTATCTAGCTCCGCXCGCTGGTACCCATCTCATGA-3’ 3’-TTAACGATAGATCGAGGCGTGCGACCATGGGTAGAGTACT-5’ Tg 5’-CCAGCGCACGACGCAtGCACGACGACCGGG-3’ 3’-GGTCGCGTGCTGCGTACGTGCTGCTGGCCC-5’

The lesions are in boldface; O for 8-oxoG, U for uracil, X for hypoxanthine, and t for thymine glycol

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8677

Figure 1 Schematic diagram of the CSB protein. The CSBE646Q point mutation in ATPase motif II is indicated. Black bars indicate putative NLS sequences

Figure 2 Capacity of mitochondrial extracts of human ﬁbroblast cell lines to incise oligonucleotide substrates with 8-oxoG, uracil, hypoxanthine to thymine glycol lesions. (a) 88.7 fmoles oligonucleotide with a single 8-oxoG (28 oligomer), hypoxanthine (hX) (40 oligomer), uracil (30 oligomer) or thymine glycol (Tg) (30 oligomer) residue was incubated with 40 mg mitochondrial extract. Incu- bations were for 4, 6, 2 or 2 h for substrates with 8-oxoG, Tg, hX or uracil lesions, respectively. Extracts were added as follows: Lane 1, no extract; lane 2, CS1AN/pc3.1CSBwt; lane 3, CS1AN/pc3.1-CSBE646Q; lane 4, CS1AN/pc3.1. (b) Data from (a) was quantiﬁed. Mean values+standard deviations are shown for assays of two samples per cell line. Oligonucleotide sequences are shown in Table 1. *(P50.005) statistically different from CS1AN/pc3.1CSBwt and CS1AN/pc3.1-CSBE646Q

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8678 Repair of other base lesions was also examined using mouse model was used to confirm the observation that mitochondrial extracts of these cell lines. Oligonucleo- CSB stimulates repair of 8-oxoG in human mitochon- tide substrates were constructed with a single uracil, drial extracts. Liver mitochondria were isolated from 6 hypoxanthine (hX) or thymine glycol (Tg) lesion (Table month old wild type and csb7/7 mice and mitochon- 1), and ability of the mitochondrial extracts to incise at drial extracts were assayed for 8-oxoG incision as the site of the lesion was measured. Figure 2a,b show described above. Five animals of each genotype were that CS1AN cells repair uracil and hX more efficiently used and the average incision efficiency (+standard than they repair 8-oxoG. In addition, expression of deviation) was calculated for each genotype. Figure 3 wild type CSB or CSB-E646Q did not alter the shows that mitochondria from CSB deficient mice have endogenous level of repair of these lesions (Figure a statistically significant (P50.05) reduction in ability 2a,b). The same result was obtained when the reactions to incize an 8-oxoG-containing substrate, but have were stopped earlier, and the very early repair rates normal ability to incize a uracil-containing substrate. were measured (data not shown). This result suggests that stimulation of DNA repair by CSB is specific for Repair of Fpg sensitive sites in mitochondria of CS-B cells 8-oxoG, and may be mediated by the mitochondrial 8- oxoG-specific glycosylase, (mtOGG1). This suggestion The repair capacity of human CS-B cells expressing is consistent with the fact that uracil, hX and Tg are wild type or mutant CSB was also assessed using an recognized and incised primarily by uracil DNA assay that measures loss of Fpg-sensitive sites in the glycosylase, 3-methyl adenine DNA glycosylase total mitochondrial genome. This assay uses the (MAG), and human homologue of endonuclease III bacterial Fpg enzyme to measure the number of lesions (hNTH1), respectively. The observation that mitochon- in mitochondrial DNA, and assesses the overall in vivo drial extracts from CS1AN cells expressing wild type repair capacity in the mitochondria of different cell CSB incize hX (19%/h) and uracil (38%/h) with a lines. DNA damage was induced in the cells using higher efficiency than 8-oxoG (5%/h) or Tg (7%/h) photoactivated methylene blue (MB), which produces may reflect differences in the specific activity of the mostly 8-oxoG base lesions (Schneider et al., 1990). glycosylases responsible for incision of each type of The initial number of Fpg-sensitive lesions induced was lesion. similar in control cells and cells expressing wild type CSB and slightly higher in CS1AN cells expressing CSB-E646Q (Table 2), and Fpg-sensitive sites were not Incision of 8-oxoG in mitochondrial extracts from CSB detected in cells not exposed to MB. deficient mice Figure 4a,b show that expression of wild type or A mouse deficient in CSB (csb 7/7) was recently ATPase-deficient CSB increases the rate of repair of constructed (van der Horst et al., 1997). This CSB Fpg-sensitive sites in mitochondrial DNA of CS1AN cells, and the difference is statistically significant (P50.005). CS1AN/pc3.1 cells repaired 34% of the Fpg-sensitive sites after 8 h and 48% after 24 h. In contrast, cells expressing wild type CSB repaired 60% of the Fpg-sensitive sites after 8 h, and 64% were removed after 24 h. This rate of repair is comparable to that seen in a normal fetal lung fibroblast cell line (Anson et al., 1998). Repair capacity was similar in cells expressing CSB-E646Q and wild type CSB. This result is consistent with the observation that CSB- E646Q complements the deficiency of CS1AN cells in incision of 8-oxoG.

Levels of OGG1 protein in mitochondrial extracts Results presented above suggest that CSB stimulates incision of 8-oxoG, the initial step of repair of this

Table 2 Initial lesion frequency in mitochondrial DNA after methy- a Figure 3 8-oxoG and uracil incision by mitochondrial extracts of lene blue treatment liver cells from wild type and CSB deﬁcient mice. 8-oxoG oligo- Cell line Initial number of lesionsb nucleotide (88.7 fmoles) containing a single 8-oxoG or uracil residue was incubated with 100 mg mitochondrial extract for 16 h at CS1AN/pc3.1 0.39 (+0.11) 328C (8-oxoG incision assay) or 40 mg extract for 1 h at 378C (ur- CS1AN/pc3.1-CSBE646Q 0.48 (+0.12) acil incision assay). Mean value+standard deviation was calcu- CS1AN/pc3.1-CSBwt 0.39 (+0.10) lated using data for three to ﬁve animals per genotype. Mean a 2 incision activity was normalized to mean wild type incision activ- Cells were treated with 100 mM methylene blue and 200 kJ/m visible ity. Note that the y-axis begins at 0.4. *(P50.05) statistically dif- light. For further details see Materials and methods. bLesions are ferent from wild type mice given as numbers of Fpg sensitive sites (FSS) (+s.d.) per 10 kb

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8679 a

Figure 5 Western blot of mitochondrial and whole cell extracts. Forty mg mitochondrial (mt) extract and 80 mg whole cell extract (WCE) was loaded on a 4 – 12% NuPage gel (Novex). Blots were probed with monoclonal anti-cdc47 (upper panel), polyclonal anti-mtOGG1 (middle panel) or monoclonal anti-cox (lower panel). Lanes 1 – 3 are mitochondrial extracts and lanes 4 – 5 are whole cell extracts prepared from the following cell lines: Lane 1: CS1AN/pc3.1-CSBwt; lane 2: CS1AN/pc3.1-CSBE646Q; lane 3: CS1AN/pc3.1; lane 4: CS1AN/pc3.1-CSBwt; lane 5: CS1AN/ pc3.1

cell extracts of CS1AN cells expressing wild type CSB (Figure 5, middle panel, lanes 4 – 5) when compared to the level in extracts from CSB deficient cells. In contrast, if the same blot was reprobed with antibody to CDC47, a nuclear protein, or cytochrome c oxidase Figure 4 Mitochondrial repair of Fpg sensitive sites in human fi- (COX), a mitochondrial protein, no difference in broblast cell lines. Fpg sensitive sites (FSS) were induced by ex- expression level was observed (Figure 5, upper and posing cells to methylene blue and light and FSS were lower panels). This result indicates that the mechanism quantified as described in Materials and methods. (a) Total DNA was isolated from the indicated cell lines 0, 8, or 24 h after by which CSB stimulates 8-oxoG repair may involve MB treatment. Control samples (Con) were not treated with transcriptional regulation of OGG1. The Western blots methylene blue or light. All samples were treated (+) or mock in Figure 5 furthermore demonstrate that the mito- treated (7) with Fpg. DNA samples were analysed by Southern chondrial extracts are free of significant nuclear blot using a probe for the mitochondrial genome. (b) FSS were contamination, as no signal for the abundant nuclear quantified and mean+standard deviation was calculated for data from three to six experiments. Two to four gels were analysed for protein, CDC47, is detected in the lanes with each experiment. *(P50.005) statistically different from CS1AN/ mitochondrial extracts (Figure 5, upper panel, lanes pc3.1CSBwt and CS1AN/pc3.1-CSBE646Q 1 – 3).

Discussion lesion. OGG1 is the glycosylase that catalyses this reaction. There are at least two splice-forms of OGG1, This study provides evidence that the CSB gene a nuclear and a mitochondrial. Thus, one possible product plays a role in repair of 8-oxoG in mitochon- explanation for our observation is that CSB stimulates drial DNA in human and mouse cells. Evidence is expression of OGG1 at the level of transcription. This presented that expression of recombinant CSB specifi- possibility is consistent with the fact that CSB cally increases incision and repair of 8-oxoG in the homologues in the SWI/SNF family regulate transcrip- mitochondria of CSB-deficient human cells, and that tion of their target genes. This idea was tested by cells from a CSB deficient mouse have reduced capacity measuring the level of mitochondrial OGG1 to incize 8-oxoG in mitochondrial DNA. This idea is (mtOGG1) in mitochondrial extracts of CSB deficient consistent with previous studies of possible biological cells and cells expressing wild type or mutant CSB by roles of CSB, which indicate involvement in the Western blot (Figure 5). The level of mtOGG1 protein response to DNA damage (Balajee et al., 1997; Dianov was significantly higher in mitochondrial extracts of et al., 1997; Evans and Bohr, 1994; Lehmann, 1982; cells that express wild type CSB or CSB-E646Q than in Ljungman and Zhang, 1996; Mayne and Lehmann, CSB deficient cells (Figure 5, middle panel, lanes 1 – 3). 1982; van Hoffen et al., 1993; Venema et al., 1990). Mitochondrial OGG1 protein was also higher in whole However, it has not previously been reported that CSB

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8680 stimulates repair of 8-oxoG or other oxidative base CS1AN cells transfected with the wild type CSB gene damage in mammalian mitochondrial DNA. (Figure 5). CSB has homology to the SWI/SNF family of The mechanism by which CSB increases the steady proteins, which share a conserved ATPase domain. state level of OGG1 is not clear. One possibility is that Previous studies indicate that ATPase-deficient mutants CSB specifically stimulates transcription of the OGG1 of CSB are sensitive to UV, undergo apoptosis more gene; OGG1 may be poorly transcribed in the absence frequently in response to UV than wild type cells, and of CSB because it is a very large transcriptional unit have reduced TCR of UV-induced pyrimidine dimers (Aburatani et al., 1997). This possibility is supported (Balajee et al., 2000; Brosh et al., 1999). However, the by RT – PCR experiments, which were performed very exact role of the ATPase domain in these processes recently by Tuo et al. (2002). In that study the basal remains unknown. The results presented here indicate transcription of OGG1 was 50% lower in CS-B cells that the ATPase domain is not required for CSB to compared with CSBwt transfected CS-B cells. Further- stimulate incision or repair of 8-oxoG in mitochondrial more, Dianov et al. (1999) have previously DNA. This conclusion is based on the observation that demonstrated that transfection of CS-B cells with the expression of CSB-E646Q, a CSB mutant completely CSB gene was associated with a 50% increase in the lacking ATPase activity, stimulates 8-oxoG incision level of hOGG1 mRNA, whereas there was no change and repair to the same extent as wild type CSB in b-actin levels. Alternatively, CSB may interact with (Figures 2 and 4). This result indicates that the CSB and stimulate OGG1 in the mitochondrion. Thus, Tuo ATPase is not essential when CSB stimulates repair of et al. (2002) recently demonstrated that there is a 8-oxoG; in contrast, the CSB ATPase is likely to be functional crosstalk between the CSB and OGG1 required for other CSB functions such as chromatin proteins in cell extracts. Evidence to support the same remodeling (Citterio et al., 2000), interaction with functional interaction in mitochondria is lacking, RNA polymerase II (Tantin et al., 1997) or TCR however, because it has not been possible to demon- (Brosh et al., 1999). strate that CSB is present in mitochondria and CSB Previous studies show that repair of 8-oxoG in the lacks a mitochondrial targeting sequence. However, the mitochondria involves exclusively BER and does not presence of CSB in mitochondria cannot be excluded involve a TCR-based mechanism (Anson et al., 1998). since CSB is likely to be a very low abundance protein The results presented here indicate that the first step in mitochondria, and it may not be detectable by the of BER, incision of 8-oxoG, is deficient in the antibodies presently available. mitochondria of cells lacking wild type CSB. Many CSB patients have a complex phenotype that glycosylases carry out the incision step of BER in a includes growth retardation, skeletal and retinal more or less lesion-specific manner. 8-oxoG is abnormalities, severe neurological deficiencies and specifically recognized and incised by OGG1, whereas premature aging (Friedberg, 1996; Nance and Berry, other glycosylases such as UDG, MAG, and hNTH1 1992). Some aspects of this phenotype overlap with the are specific for other base lesions. However, CSB phenotype associated with mitochondrial dysfunction specifically stimulates repair of 8-oxoG in mitochon- (i.e., neurological disease and dysfunction in heart, drial DNA, but does not stimulate repair of uracil, skeletal muscle and kidneys (Wallace et al., 1998)). hX or Tg (Figures 2 and 3). CSB stimulation was This study provides evidence that deficiency in CSB specific for repair of 8-oxoG in both human and leads to a defect in mitochondrial DNA repair of the mouse cells, suggesting that this observation is not an oxidative lesion 8-oxoG. Thus, it is possible that CSB artefact of the experimental system, but reflects the cells accumulate mutations in mitochondrial DNA and specificity of CSB function in mitochondrial DNA develop mitochondrial dysfunction that contributes to repair. the phenotype and progression of disease in CSB Recent studies demonstrate that OGG1 is the patients. glycosylase that initiates repair of 8-oxoG by BER. Klungland et al. (1999) studied OGG1 knockout mice and demonstrated that these animals accumulate more 8-oxoG in genomic DNA than wild type mice, and that Materials and methods nuclear extracts from ogg17/7 mouse cells remove 8- oxoG at a slower rate than wild type cells. Similar Cells and cell culture conditions defects were recently demonstrated in mitochondrial The SV40-transformed human fibroblast cell line DNA and mitochondrial extracts (de Souza-Pinto et CS1AN.S3.G2 belongs to CS complementation group B al., 2001). In addition, Dobson et al. (2000) showed and was described previously (Mayne et al., 1982, 1984). that mitochondrial repair of oxidative DNA damage CS1AN.S3.G2 cells were stably transfected with pcDNA3.1 increases in cells that express recombinant OGG1 with (Invitrogen, abbreviated as pc3.1) vector only (CS1AN/ pc3.1), pc3.1 vector containing the wild type human CSB a mitochondrial targeting sequence. These data indicate gene (CS1AN/pc3.1-CSBwt), and pc3.1 vector containing the that OGG1 is essential for 8-oxoG repair in eukaryotic CSB-E646Q mutated gene (CS1AN7pc3.1-CSBE646Q). These cells, and raise the possibility that CSB influences the cell lines were described previously (Selzer et al., 2002). Cells rate of 8-oxoG repair by regulating OGG1 expression were grown in minimal essential medium (MEM) supple- or activity. This hypothesis is supported by the mented with 15% fetal bovine serum and 400 mg/ml geneticin observation that mtOGG1 increases markedly in (Invitrogen, Denmark).

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8681 Mice Induction of mitochondrial oxidative DNA base damage (8-oxoG) in cultured cells Mice used in this study are strain C57B16 with or without a targeted mutation of the CSB gene (van der Horst et al., Oxidative damage was introduced into mitochondrial DNA 1997). Animals were housed under standard conditions and as described by Anson et al. (1998). Briefly, exponentially euthanized by cervical dislocation as required. Mouse livers growing cells were incubated for 1 h with 100 mM Methylene were isolated immediately after euthasia, washed free of Blue(MB) in PBS containing 0.5 mM MgCl2,1mM CaCl2, blood with M-SHE buffer (0.21 M mannitol, 0.07 M sucrose, and 1% glucose. Cells were transferred to PBS, exposed to 2 10 mM HEPES (pH 7.4), 1 mM EDTA, 1 mM EGTA, visible light (100 kJ/m ), washed twice with PBS and lysed 0.15 mM spermine, 0.75 mM spermidine, 5 mM DTT, 2 mM immediately or allowed to repair in growth medium contain- benzamidine-HCl, 2 mg/ml leupeptin) and mouse liver ing 10 mM bromodeoxyuridine and 1 mM fluorodeoxyuridine. mitochondria (MLM) extracts were prepared as described All manipulations were performed in dim blue light. elsewhere (Souza-Pinto et al., 1999). Gene specific repair in mitochondrial DNA Isolation of mitochondria from cells in culture The gene specific repair assay was performed essentially as Mitochondria were isolated from cultured human fibroblasts described by Anson et al. (1998). Briefly, cells were lysed 0, 8 using a combination of differential centrifugation and Percoll or 24 h after exposure to light. DNA was isolated, digested gradient centrifugation in a protocol modified from Croteau with restriction endonuclease PvuII, and replicated DNA was et al. (1997). Briefly, actively growing cells were resuspended separated from unreplicated DNA by neutral CsCl2 density and washed twice with PBS, pelleted at 500 g and the cell gradient centrifugation. Parental unreplicated DNA was pellet resuspended in M-SHE buffer. The cell suspension was treated with Fpg, which nicks at sites of 8-oxoG lesions in homogenized using a glass-to-teflon Dounce homogenizer, DNA. Fpg-treated and mock-treated DNA samples (1 mg) and the homogenate was differentially centrifuged and for each time point were separated under alkaline conditions subjected to density gradient centrifugation in a Percoll (30 mM NaOH, 1 mM EDTA) on a 0.6% agarose gel. DNA gradient. Purified mitochondria were stored as pellets at – was transferred to a Hybond N+ membrane (Amersham 808C. Pharmacia Biotech) using standard blotting procedures. The mitochondrial DNA probe, pCRII (previously described by (Anson et al., 1998)), was labeled using a random prime Oligonucleotides and mitochondrial probe labeling kit (Amersham Pharmacia Biotech) and hybridized Oligonucleotide substrates were purchased from Midland to the membrane at 688C overnight. The membrane was Certified Reagent Co. (Midland, TX, USA). Oligonucleotide washed under stringent conditions. The blots were visualized sequences are shown in Table 1. Oligonucleotides were 5’ by Molecular Imager FX and quantified using QuantityOne end-labeled using T4 polynucleotide kinase and [g32-P]ATP. software (Bio-Rad, CA, USA). The ratio of Fpg sensitive Unincorporated radioactivity was removed with G25 spin DNA to the total DNA in the reaction was calcuated. The columns. pCRII was used as a mitochondrial DNA probe as frequency of Fpg sensitive sites (FSS) was determined by the described previously (Anson et al., 1998). Poisson distribution. Gene-specific repair was expressed as percent repair activity as described previously (Bohr et al., 1988). Measurement of 8-oxoG incision activities in mitochondrial extracts Western analysis Glycosylase/AP-endonuclease activity was measured using an oligonucleotide incision assay as previously described (Souza- Forty to eight mg of mitochondrial protein or whole cell Pinto et al., 1999). Briefly, intact mitochondria were resus- extracts (prepared according to Manley et al. (1983) from pended in buffer (20 mM HEPES-KOH (pH 7.6), 1 mM CS1AN/pc3.1, CS1AN/pc3.1-CSBE646Q, and CS1AN/pc3.1- EDTA, 2 mM DTT, 300 mM KCl, 5% glycerol, and 0.05% CSBwt cells were separated on 4 – 12% NuPage gels (Novex) Triton X-100), diluted to 15 mg/ml protein with the same buffer and transfered to PVDF membranes (Novex) by electroblot- lacking Triton X-100 and adjusted to 100 mM KCl. Incision ting in transfer buffer (25 mM Bicine, 25 mM Bis-Tris pH 7.2, reactions (20 ml) contained 20 mM HEPES-KOH (pH 7.6), 1mM EDTA, 10% methanol) for 1 h at 30 V. The membrane 5mM EDTA, 5 mM DTT, 75 mM KCl, 5% glycerol, 0.1 mg/ml was blocked for 16 h at 48C in 5% non fat dry milk (BioRad) 32 BSA, 88.7 fmoles of P-labeled duplex oligonucleotide, and in TBST (20 mM Tris-HCl pH 7.2, 137 mM NaCl, 0.1% mitochondrial protein as indicated. In order to increase incision Tween-20). Blots were probed with primary antibody diluted efficiency at 8-oxoG, reactions with mitochondria from human in TBST; primary antibody was mouse monoclonal anti- cells in culture were supplemented with 0.5 mM MgCl2. cdc47 (MS-862-P0; NeoMarkers), rabbit polyclonal anti- Reactions were incubated at 328C (mouse liver mitochondria) mtOGG1 (ab6491, Novus Biologicals) or mouse monoclonal or 378C (human mitochondria) for 2 – 16 h as indicated, anti-cytochrome oxidase IV (subunit V) (A6456, Molecular terminated by addition of 0.8 ml 5 mg/ml Proteinase K and Probes). Detection was performed with ECL+Plus1 (Amer- 0.8 ml 10% SDS and incubated at 558C for 15 min. As a control sham-Pharmacia Biotech). reaction 8-oxoG containing oligonucleotides were treated with 5 – 10 ng Escherichia coli formamidopyrimidine DNA glycosylase (Fpg) for 1 h at 378C (data not shown). The DNA was ethanol-precipitated and analysed in a denaturing 20% polyacrylamide gel containing 7 M urea. The gels were Abbreviations visualized using a Molecular Dynamics PhosphorImager, and 4-NQO, 4-Nitroquinoline-1-oxide; 8-oxoG, 7,8-dihydroxy- quantified using ImageQuant NT software. Incision activity guanine; AP, apurinic/apyrimidinic; BER, base excision was calculated from the ratio of damage-specific cleavage repair; CS, Cockayne Syndrome; CSB, Cockayne Syndrome product to the total product and substrate in the reaction. group B; Fpg, formamidopyrimidine DNA glycosylase; FSS,

Oncogene Deficient mitochondrial repair of 8-oxoG in CSB T Stevnsner et al 8682 Fpg sensitive sites; hNTH1, human homologue of endonu- Acknowledgments clease III; hX, hypoxanthine; MB, Methylene blue; NA-AAF, Tanja Thybo helped prepare the mouse liver mitochondria. N-acetoxy-2-acetylamino-ﬂuorene; MLM, mouse liver mito- Ulla Henriksen and Alfred May provided technical chondria; MAG, 3-methyl adenine DNA glycosylase; mt, assistance. The project was supported by Danish Research mitochondrial; NER, nucleotide excision repair; OGG1, 8- Council, NovoNordic Foundation, The Frænkel Founda- oxoguanine DNA glycosylase; ROS, reactive oxygen species; tion, The Neye Foundation, The Foundation of 17-12- TCR, transcription coupled repair; Tg, Thymine glycol; 1981, The Netherlands Organization for Scientiﬁc UDG, uracil DNA glycosylase; UV, ultraviolet; WCE, whole Research, and The European Community (QLK6-CT- cell extract 1999-02002).

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