P53 Functions in the Incorporation Step in DNA Base Excision Repair in Mouse Liver Mitochondria

Home , AP endonuclease, Base excision repair, Uracil

Oncogene (2004) 23, 6559–6568 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc p53 functions in the incorporation step in DNA base excision repair in mouse liver mitochondria

Nadja C de Souza-Pinto1, Curtis C Harris2 and Vilhelm A Bohr*,1

1Laboratory of Molecular Gerontology, NIA-IRP, National Institutes of Health, Baltimore, MD 21224, USA; 2Laboratory of Human Carcinogenesis, CTR, NCI, National Institutes of Health, Bethesda, MD 20892, USA

The tumor suppressor p53 protein stimulates nuclear base direct participation of p53 in base excision repair (BER) excision repair (BER) in vitro. In response to certain has been reported. BER is the major DNA repair cellular stresses, p53 translocates to mitochondria, where pathway for the processing of small base modifications, it can trigger an apoptotic response. However, a potential such as oxidative and alkylation damage. Offer et al. role for p53 in modulating mitochondrial DNA repair has (1999)demonstrated that p53 expression augments not yet been examined. In this study, we show that p53 apurinic/apyrimidinic (AP)site-induced DNA repair also modulates mitochondrial BER. Uracil-initiated BER synthesis incorporation. This stimulation was found incorporation, which measures flux through the entire to be independent of the p53 transcriptional activity BER pathway, was lower in mitochondrial extracts from because a trans-activation-deficient p53 mutant was nonstressed p53 knockout mice than in wild type. The more efficient in stimulating BER than wild-type p53 addition of recombinant p53 complemented the BER (Offer et al., 2001). The mechanism by which p53 incorporation in p53 knockout extracts and stimulated stimulates BER is still unclear. A direct interaction of BER in wt extracts. The activities of three major p53 with APE 1 (Meira et al., 1997; Gaiddon et al., mitochondrial DNA glycosylases were similar in extracts 1999)and with DNA polymerase (pol) b, and the from wild-type and knockout animals. Likewise, AP stabilization of pol b binding to the AP site by p53 have endonuclease activity was unaffected by the absence of been documented (Zhou et al., 2001). Moreover, Seo p53. Gel shift experiments with recombinant p53 demon- et al. (2002)demonstrated a deficient pol b expression in strated that p53 did not bind to the uracil-containing p53-deficent cell lines. substrate used in the repair assay. Polymerase c gap-filing Recent results also suggest a role for p53 in activity was less efficient in p53 knockout extracts, but it controlling mitochondrial function and response to was complemented with the addition of recombinant p53. stress (Moll and Zaika, 2001). After various death Thus, we conclude that p53 may participate in mtBER by stimuli, a fraction of the induced p53 protein trans- stimulating the repair synthesis incorporation step. locates to mitochondria, where it elicits a series of Oncogene (2004) 23, 6559–6568. doi:10.1038/sj.onc.1207874 responses that can ultimately lead to cell death Published online 21 June 2004 (Marchenko et al., 2000; Dumont et al., 2003; Mihara et al., 2003). However, it is unknown whether p53 plays Keywords: p53; mitochondrial DNA; DNA repair; any role in DNA repair in mitochondria, and whether BER; uracil the protein is even present in the mitochondria in the absence of any stress. Mammalian mitochondria are very proficient at Introduction removing small base modifications through the BER pathway (for a review, see Bohr et al., 2002). BER is The tumor suppressor protein p53 plays a central role in initiated by a DNA glycosylase that specifically removes controlling cell fate after various stresses (Vogelstein the damaged base, generating an abasic (AP)site. The et al., 2000). p53 protein is involved in many aspects of AP site is processed by an AP endonuclease, or by the the cellular response to DNA damage and its expression glycosylase-associated AP lyase activity. This activity and protein stability are both enhanced after damage. In introduces a single DNA strand break at the site of addition to its primary cellular function as a transcrip- damage. After the processing of the ends and the tion activator, p53 is directly involved in DNA repair, insertion of a new nucleotide by DNA polymerase most notably nucleotide excision repair (Evans et al., gamma (pol g), the nick is sealed by DNA ligase, 1993; Wang et al., 1995; Hanawalt, 2002). Recently, a regenerating the parental DNA. Several of the enzymes involved in mitochondrial BER have been characterized. In many cases, the same gene encodes for both the *Correspondence: VA Bohr, Laboratory of Molecular Gerontology, mitochondrial and the nuclear enzyme, as it has been Box 1, National Institute of Aging, GRC, NIH; 5600 Nathon Shock Dr., Baltimore, MD 21224, USA; E-mail: [email protected] demonstrated for uracil DNA glycosylase (UDG) Received 20 February 2004; revised 16 April 2004; accepted 3 May 2004; (Nilsen et al., 1997), oxoguanine DNA glycosylase published online 21 June 2004 (OGG1)(Souza-Pinto et al., 2001)and DNA ligase III p53 in mtDNA repair NC de Souza-Pinto et al 6560 (Lakshmipathy and Campbell, 1999). Although this demonstrates that our extracts were free of nuclear pathway has been reconstituted in vitro (Stierum et al., contamination. 1999a), it is unclear how mitochondrial BER (mtBER) is A role for p53 in nuclear BER has been previously regulated in vivo. demonstrated using human and mouse cell lines. In Since p53 modulates nuclear BER, and mtBER order to investigate the involvement of p53 in mtBER, utilizes a similar set of proteins to that of nuclear we isolated mitochondria and nuclei from livers of BER, we asked whether basal p53 also modulates BER wild-type, p53 heterozygote (p53À/ þ )and p53 knockout in mitochondria. To address this question, we compared (p53À/À)mice. Base excision repair was analysed as the DNA repair activities in mitochondrial and nuclear incorporation of a radiolabeled dCTP nucleotide into an extracts obtained from unstressed mice with basal p53 unlabeled 30-mer double-strand substrate containing a levels and from heterozygous (p53À/ þ )and knockout U/G base pair. This assay measure the flux through the (p53À/À)animals. Our results suggest that the glycosylase entire BER pathway, since the incorporation of radio- and AP-endonuclease steps of BER are not directly activity into the full-length oligonucleotide requires affected by p53 status. On the other hand, the repair the combined activities of a glycosylase, an AP- synthesis incorporation step, which is catalysed in endonuclease, a DNA polymerase and a DNA ligase. mitochondria by pol g, was lower in p53À/À extracts. The incubation of a uracil-containing unlabeled 30-mer This defect was complemented by the addition of oligonucleotide (U30)with mouse liver nuclear extracts recombinant p53. Altogether, these results suggest that (MLNE)resulted in the appearance of a 30-mer p53 modulates mtBER through the stimulation of the radioactive band, indicating the incorporation of 32P- nucleotide incorporation step. dCTP in place of the uracil damage (Figure 2, panel a). A small amount of radioactivity was incorporated into the control oligonucleotide, most likely due to some unspecific nick-directed DNA synthesis. For quantifica- Results tion, the incorporation with control oligonucleotide was subtracted from the signals obtained with the uracil- One major concern when investigating mitochondrial containing oligonucleotide. The intensity of the 30-mer DNA repair is the possibility that the mitochondrial band increased in a linear fashion with the amount of extracts are contaminated with nuclear DNA repair protein added to the assays and with incubation time enzymes. In order to ascertain that our mouse liver (not shown). In the MLNE from p53À/À mice, we found mitochondrial extracts were free of nuclear contamina- 40% less incorporation than in wt (Figure 2a), suggest- tion, we analysed for the presence of Lamin B2 in ing that p53 directly participates in BER in the mouse mitochondrial extracts, by Western blots. Lamin B2 was liver nuclei. BER incorporation in the p53À/ þ hetero- chosen because it is a very abundant nuclear protein, zygote extracts was similar to wt, and these extracts thus and thus, we would be able to detect even relatively contained sufficient p53 to support BER. small amounts of nuclear contamination in the mito- The incubation of U30 oligonucleotides, but not C30, chondrial fractions. Antibodies against cytochrome with mouse liver mitochondrial extracts (MLME) oxidase subunit IV (COX IV)were used as marker for resulted in the appearance of a 12-mer radioactive band mitochondrial content. Figure 1 shows a typical blot (Figure 2b). This band corresponds to the unligated from nuclear (lanes 1–2)and mitochondrial (lanes 3–4) intermediate generated after uracil removal and incor- extracts from wild-type and p53À/À animals. The absence poration of the new nucleotide. We have previously of the lamin B2 signal in the mitochondrial extracts observed that MLME, in our hands, have week DNA ligase activity and thus accumulate the unligated intermediate (Stuart et al., 2004). It is important to point out that the 12-mer intermediate is a ligatable species, since addition of T4DNA ligase to the reactions converts the 12-mer intermediate into the full-length 30-mer product (not shown). Interestingly, MLME from p53À/ þ and p53À/À showed lower incorporation activity than wild-type extracts, by 10 and 21%, respectively. Although the differences were not statistically significant, the gradual decrease over heterozygous to homozygous does suggest a role for p53 in mitochondrial BER. We then determined whether the decreased BER activity in extracts from p53À/À mice was related to the absence of the p53 protein or to an indirect effect of Figure 1 Western blot analysis of mouse liver extracts: 10 mgof the p53 deficiency. We performed uracil-initiated BER mouse liver nuclear (MLNE)or mitochondrial (MLME)extracts assays with nuclear and mitochondrial extracts from wt were separated by SDS–PAGE. The proteins were transferred to a À/À PVDF membrane and blotted with monoclonal anti-Lamin B2 and p53 , with or without the addition of recombinant (NovoCastra)and monoclonal anti-cytochrome oxidase subunit IV p53 protein (Figure 3, panels a and b). In both cases, (COX IV)(Molecular Probes) 0.5 mg of recombinant p53 fully complemented the

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6561 contains a U/G mismatch, and thus might affect BER activity through binding to the DNA substrate. Re- combinant full-length p53 was incubated with one of the following oligonucleotides: a 30-mer oligonucleotide containing the partial p53 consensus binding sequence (CATG)(p53C),the U30 oligonucleotide and oligonucleotides in which a damaged base (either uracil (p53U) or 8-oxoguanine (p53OG)) was introduced within the p53-binding site. Recombinant p53 efficiently shifted the p53C oligonucleotide but not the U30 (Figure 4, lanes 1 and 2), indicating that the repair substrate was not bound by p53 in vitro. The presence of a damaged base in the p53 consensus sequence diminished DNA binding by p53 (Figure 4, lanes 3–4). These results indicate that the complementation of uracil-initiated mtBER activity observed after the addition of recombinant p53 was not due to p53 binding to the substrate, but rather a direct effect of the protein in one or more enzymatic steps of the BER pathway. The first step in BER is catalysed by a DNA glycosylase, which recognizes the damaged base. Each DNA glycosylase recognizes a defined set of modified bases, and thus these enzymes provide the specificity of the repair process (Krokan et al., 1997). In mitochondria, three major DNA glycosylases have been characterized: mtUDG, which catalyses the removal of uracil (Domena and Mosbaugh, 1985); mtOGG1, which removes oxidized purines, most notably 8-oxoG (Souza- Pinto et al., 2001); and mtNTH1, which recognizes oxidized pyrimidines (Karahalil et al., 2003). We measured the activities of each of these enzymes in mitochondrial and nuclear extracts from wt, p53À/ þ and p53À/À mice to investigate whether p53 levels modulate activity of those enzymes. DNA glycosylase activity was measured as incision of a radiolabeled single lesion- containing double-strand oligonucleotide. To measure Figure 2 Uracil-initiated repair synthesis incorporation in mito- UDG activity, we used an oligonucleotide containing a chondrial and nuclear mouse liver extracts: Repair incorporation single uracil (U30, Table 1). For OGG1 and endonu- reactions were carried out with undamaged (C30, lanes 2–4)or uracil-containing (U30, lanes 5–7)30-mer double-strand oligonu- clease III homologue 1 (NTH1), substrates containing a cleotides, with 50 mg of mitochondrial (a)or nuclear ( b)extracts for single 8-oxoG or 5-OHC were used, respectively (OG30 4 or 2 h, respectively, at 321C. The DNA was precipitated and and OC30, Table 1). In mitochondrial extracts, UDG resolved in a 20% PAGE and the band intensities were quantified. activity is many-fold higher than OGG1 and NTH1. Relative incorporation was calculated relative to the incorporation obtained with wild-type extracts incubated with uracil-containing However, no differences in any of the three enzymes À/ þ oligo. Radioactivity present in control lanes was subtracted from activities were observed in extracts from wt, p53 or values obtained with damaged substrate. Results presented are the p53À/À animals (Figure 5, panel a). Similarly, UDG mean7standard deviation of three animals per genotype activity was higher than OGG1 and NTH1 in the nuclear extracts. No differences in the glycosylase activities were observed among the different genotypes deficient BER in the p53À/À extracts (see bar graphs). in nuclear extracts either (Figure 5, panel b). These Additional p53, up to 1 mg, did not enhance BER results suggest that p53 does not directly act in the activity further. The complementation of the uracil- glycosylase step of BER initiated by UDG, OGG1 or initiated BER deficiency in mitochondrial extracts from NTH1, since absence of p53 did not change activity of p53À/À mice was dependent on the integrity of the p53 any of those enzymes. protein, since 0.5 mg of heat-denatured (5 min at 951C, The removal of the damage base by the DNA followed by 10 min in ice)failed to complement the glycosylase yields the generation of an AP site. AP sites defect (Figure 3c). Similarly, addition of excess BSA are mostly processed by an AP endonuclease, although (1 mg)failed to stimulate mitochondrial BER. some glycosylases, such as OGG1 and NTH1, posses an Since p53 is a DNA-binding protein that efficiently associated AP lyase activity that can also catalyse the binds some DNA mismatches (Foord et al., 1991; Lee cleavage of the phosphate backbone. This step leads to et al., 1995; Degtyareva et al., 2001), we tested whether the formation of a single-strand break and creates the p53 could bind to our particular BER substrate, which substrate for the incorporation step, in which the new

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6562

Figure 3 p53 stimulation of BER in mitochondrial and nuclear mouse liver extracts: 50 mg of nuclear (a)or mitochondrial ( b)extracts from wild type (lanes 1–4)or p53 knockout (lanes 5–8)were incubated with C30 (lanes 1 and 5)or U30 (lanes 2–4 and 6–8) oligonucleotides for 2 h at 321C, in the absence or presence of 500 ng or 1 mg of recombinant p53. (c)50 mg of wt (lanes 1 and 2)or p53À/À (lanes 3–6)mitochondrial extracts were incubated with C30 (lane 1)or U30 (lanes 3–6)oligonucleotides for 2 h at 32 1C, with the following additions: 0.5 mg of recombinant p53 (lane 4), 0.5 mg of heat-denatured p53 (lane 5)or 1 mg of BSA (lane 6). The graphs present the mean7standard deviation of two animals per genotype, each assayed twice

nucleotide is added to the DNA. Thus, we tested cleavage but is resistant to b-elimination catalysed by whether AP endonuclease activity varied in mitochon- AP lyases (Wilson et al., 1995). Mitochondrial extracts drial extracts from wt, p53À/ þ and p53À/À mice. AP from animals of the three different genotypes have very endonuclease activity was monitored as incision of an similar THF incision activity (Figure 6), indicating that oligonucleotide containing a tetrahydrofuran AP ana- basal mitochondrial AP endonuclease does not change log, which is a model substrate for AP endonuclease with p53 status.

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6563 extracts (not shown). Together, these results suggest a direct action of p53 on the nucleotide incorporation activity of pol g.

Discussion

The various roles of p53 in the cellular responses to DNA damage are still not clear. Although not required for BER (Sobol et al., 2003), p53 has been shown to stimulate nuclear BER in vitro (Zhou et al., 2001), in cells in culture (Offer et al., 1999)and now in mouse liver (this work). Ours is the first study on the direct role of p53 on mitochondrial BER. Mitochondrial BER resembles nuclear BER, and many of the repair enzymes in both compartments are encoded by the same genes. Thus, we hypothesized that p53 modulates mitochondrial BER in a similar fashion as it does nuclear BER. To test this hypothesis, we compared BER activities in liver mitochondrial and nuclear extract from wild-type, p53À/ þ and p53À/À mice. We found that uracil-initiated BER activity is lower in mitochondria and nuclei from p53 knockout mice. The BER impairment is related to the absence of the p53 protein itself, as supported by two lines of experimental evidence. Firstly, addition of recombinant p53 restored BER activity to wild-type levels both in mitochondrial and nuclear extracts. Secondly, the p53 complementa- Figure 4 Binding of p53 to the oligonucleotide substrates: tion depended on the integrity of the protein, since heat- 4 pmoles of recombinant p53 or BSA were incubated with denatured p53 or excess BSA did not complement the 300 fmoles of 32P-labeled double-strand oligonucleotides (Table 1). BER defect in mitochondrial extracts from the p53 The protein–DNA complexes were detected as a shift in the oligonucleotide’s mobility in a 5% PAGE (a).(b)Quantification of knockout animals. Taken together, these results suggest % of shifted substrate. Results are average 7 standard deviation of that p53 functions in one or more steps in the BER three separate experiments pathway in mitochondria. Our observations with mouse liver nuclear extracts are also in agreement with the previously published observation that p53-defficient The results above suggested that the incorporation mouse cell lines have decreased AP-initiated BER step might be the target for p53 modulation of activity (Offer et al., 2001). mitochondrial BER. We thus investigated pol g activity We investigated whether the role of p53 in mtBER in mitochondrial extracts from wt and p53À/À mice. Pol g was at the DNA glycosylase step. DNA glycosylases activity was measured using a gap-filling assay, in which initiate BER by recognizing and removing the damaged a 34-mer substrate (Table 1)containing a one-nucleotide base. However, we found that p53 levels did not affect gap is incubated with the extracts in presence of a the activities of the three major glycosylases so far radioactivity nucleotide. Polymerase activity is mea- characterized in mitochondria: mtUDG, mtOGG1 and sured as the amount of radioactivity incorporated in the mtNTH1. Similarly, UDG, OGG1 and NTH1 activities 50 nucleotide. Incubation of the GAP oligonucleotide in nuclear extracts from p53À/ þ and p53À/À were no with increasing amounts of wt mitochondrial extracts different from wt extracts. Although OGG1 and NTH1 yields a dose-dependent increase in the amount of activities were significantly lower than that of UDG, radioactivity incorporated (Figure 7a). Incubation of particularly in mitochondrial extracts, those levels of the substrate with mitochondrial extracts from p53À/À incision activity were still sufficient to detect any mice results in less incorporation of radioactivity possible defects in absence of p53. We have previously compared to the wt, suggesting that pol g activity is observed significant changes in OGG1 activity with age, lower in mitochondria from p53 knockout mice. To even with the low incision levels observed in in vitro ascertain that the decreased pol g activity was related to assays (Souza-Pinto et al., 1999, Souza-Pinto et al., the absence of p53 itself, and not to a secondary effect of 2001). These results demonstrate that the glycosylase p53 knockout, we added recombinant p53 to the gap step is not directly modulated by p53 in mitochondria or filing assays (Figure 7b). In all, 0.25 mg of p53 fully nuclei, under basal, nonstressed conditions. Recently, complemented the defect in pol g gap-filling activity. Zurer et al. (2004)demonstrated that 3-mehyladenine Interestingly, addition of more p53 stimulated pol g even DNA glycosylase was modulated by p53, but this further, both in the p53À/À and in wt mitochondrial modulation depended on genotoxic stress. Thus, it is

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6564 Table 1 Oligonucleotides used in this study Oligo C30 ATA TAC CGC GGC CGG CCG ATC AAG CTT ATT TAT ATG GCG CCG GCC GGC TAG TTC GAA TAA

U30 ATA TAC CGC GGU CGG CCG ATC AAG CTT ATT TAT ATG GCG CCG GCC GGC TAG TTC GAA TAA

OG30 ATA TAC CGC GOGC CGG CCG ATC AAG CTT ATT TAT ATG GCG CCG GCC GGC TAG TTC GAA TAA

OC30 ATA TAC CGC GGOC CGG CCG ATC AAG CTT ATT TAT ATG GCG CCG GCC GGC TAG TTC GAA TAA

p53C TAC AGA ACA TGT CTA AGC ATG CTG GGG ATG TCT TGT ACA GAT TCG TAC GAC CCC

p53U TAC AGA AUA TGT CTA AGC ATG CTG GGG ATG TCT TGT ACA GAT TCG TAC GAC CCC

p53OG TAC AGA ACA TOGT CTA AGC ATG CTG GGG ATG TCT TGT ACA GAT TCG TAC GAC CCC

THF CAA GAC CTF GGC GTC C GTT CTG GAA CCG CAG G

GAP CTG CAG CTG ATG CGC _GT ACG GAT CCC CGG GTA C GAC GTC GAC TAC GCG GCA TGC CTA GGG GCC CAT G

The important features are underlined. U, uracil; OG, 8-oxoG; OC, 5-OHC; F, tetrahydrofuran

possible that p53 stimulation of the glycosylase step drial extracts from p53-deficient mice. Thus, it is very represents only a small component of the p53 modula- likely that the lower activity of uracil-initiated BER tion of BER, under specific stress conditions. incorporation in the p53KO extracts is caused by the The second step in BER is catalysed by an AP- slower nucleotide incorporation activity of pol g in the endonuclease. APE1, the major human AP endonu- absence of p53. Additional support for this conclusion clease, has been shown to interact with and activate p53 comes from the observation that recombinant p53 not (for a review, see (Evans et al., 2000)). However, AP only complemented the lower pol g activity in p53À/À, endonuclease activity, measured as incision of a THF- but further stimulated pol g, in a dose-dependent containing substrate, did not differ in mitochondrial manner. These results suggest a direct effect of p53 on extracts from p53 heterozygous and knockout mice. pol g enzymatic activity, and also indicate that the These results suggest that, under nonstress conditions, deficient BER activity in mitochondria from p53 p53 does not modulate the AP endonuclease step of knockout mice is due exclusively to the lack of p53 BER in mitochondria. These results are in agreement itself. Although a direct interaction between p53 and pol with the observations reported by Seo et al. (2002), who g has not yet been demonstrated, pol g is stimulated by found no changes in AP endonuclease activity in p53- PCNA (Taguchi et al., 1995), which directly interacts null human whole-cell extracts. with p53. Taken together, our results suggest that basal levels of In conclusion, our results indicate that p53 may play a p53 may function in the nucleotide incorporation/ role in modulating mitochondrial DNA repair. The termini-processing step in mitochondrial BER. This observations that addition of p53 stimulates repair would be consistent with our observations that uracil- synthesis incorporation and pol g activity suggests that initiated BER activity is lower in p53À/À mitochondrial p53 may be one component of a stress response pathway extracts, but that mtUDG and AP endonuclease that involves upregulation of mitochondrial repair, and activities are unchanged. In the nuclei, both incorpora- thus plays a direct role in maintaining mitochondrial tion and termini processing activities are catalysed by DNA stability. In support of this notion, Habano et al. pol b (Allinson et al., 2001), while in mitochondria they (2000)found a direct association between alterations in are catalysed by pol g (Longley et al., 1998; Stierum the p53 gene and microsatellite instability in both the et al., 1999b). The observation that p53 directly interacts nuclear and the mitochondrial genomes. We (Hofseth with pol b in vitro (Zhou et al., 2001), together with et al., 2003)and others (Yamada and Farber, 2002)have reported lower levels of pol b in p53-null human cells shown that an adaptive imbalance in BER enzymes (Seo et al., 2002)suggest that the polymerase step is can cause microsatellite instability, including during likely the step in which p53 exerts it modulator effect in ulcerative colitis, a chronic inflammatory disease. In the nucleus. Our results suggest that a similar mechan- addition, nitric oxide, which is produced in mitochon- ism may be in effect in mitochondria. We found that pol dria at high levels, can increase 3-methyl Adenine g nucleotide incorporation activity is lower in mitochon- glycosylase activity and cause deamination of cytosine

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6565

Figure 6 AP endonuclease activity in mouse liver mitochondrial extracts: 1 mg of mitochondrial extracts from p53 wild-type (wt), heterozygous ( þ /À)or knockout ( À/À)mice were incubated with 100 fmoles of THF oligonucleotide for 15 min at 321C. The samples were resolved as described. Percent incision was calculated as the amount of radioactivity in the product band over the total radioactivity in the lane. Results presented are the mean7standard deviation of three animals per genotype. Each mitochondrial preparation was assayed twice

Institute, NIH (Frederick, MD, USA)and killed within 2 days of their arrival at the Gerontology Research Center (GRC). All experiments were approved by the GRC Animal Care and Use Committee (ACUC)and performed in Figure 5 DNA glycosylase activities in mitochondrial and nuclear accordance with the Guidelines for the Use and Care of mouse liver extracts: 100 mg of mitochondrial (MLME)( a)or 10 mg Laboratory Animals (NIH Publication 85–23). of nuclear (MLNE)( b)extracts from p53 wild-type (wt), heterozygous ( þ /À)or knockout ( À/À)mice were incubated with Oligonucleotides 1 ng of oligonucleotides containing a single uracil, 8-oxoG or 5- OHC for 6 h at 321C. The samples were resolved as described. Oligonucleotides used in this study were synthesized by Percent incision was calculated as the amount of radioactivity in Midland Certified Reagents Co. (Midland, TX, USA), with the product band over the total radioactivity in the lane. Results exception of OC30 and THF, which were kindly provided by 7 presented are the mean standard deviation of three animals per John Essigman, MIT, Cambridge, MA, USA. The sequence of genotype. Each mitochondrial preparation was assayed twice each oligonucleotide is presented in Table 1. For incision reactions and EMSA assays, the substrates were 50-32P-labeled by incubating with g32P-ATP in the presence of T4 Poly- nucleotide kinase. After the kinase reaction, unincorporated by nitrosative stress to form uracil (Krokan et al., 2002). 32P-ATP was separated from the oligonucleotides with a G25 Thus, the appropriate coordination of BER enzymes, desalting column. The labeled oligonucleotides were then and its modulation by p53, may be fundamental in annealed to the complementary strand (fivefold excess)in the maintaining genomic stability in mitochondria after presence of 100 mM KCl, by briefly incubating at 901C and genotoxic and oxidative stress. allowing them to slowly cool down to room temperature. For repair synthesis incorporation reactions, unlabeled substrates were annealed as above. Materials and methods Isolation of liver mitochondria and preparation of extracts Materials Mouse liver mitochondria (MLM)and nuclei (MLN)were Protease inhibitors were from Roche. Isotopes were from isolated using a combination of differential centrifugation NEN Life Science Products. G25 spin columns and Percoll and Percoll gradient centrifugation, as previously described were from Pharmacia. T4 polynucleotide kinase and T4 DNA (Souza-Pinto et al., 1999). After isolation, the mitochondrial ligase were from Stratagene. Recombinant p53 protein was fraction was suspended in 20 mM HEPES (pH 7.6), 1 mM obtained from Santa Cruz Biotech or kindly supplied by Dr EDTA, 5% glycerol, 0.015% Triton X-100, 5 mM dithio- Kent Soe, Institute of Molecular Biotechnology, Germany. All threitol and 300 mM KCl with the following protease inhibitors other reagents were ACS grade. added just before use: 1 mg/ml aprotinin, 1 mg/ml pepstatin A, 1 mg/ml chymostatin A, 2 mg/ml leupeptin, 2 mM benzami- dine HCl, 1 mM phenylmethylsulfonyl fluoride and 1 mM Animals E-64. Mitochondria were solubilized by adding sterile 10% Wild-type, p53 heterozygous and knockout mice, in a C57Bl Triton X-100 to a final concentration of 0.5% and clarified by background, were obtained from the National Cancer centrifugation for 1 h at 130 000 g. The resulting supernatant

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6566 Oligonucleotide incision assays UDG activity was measured in a 20 ml reaction containing 70 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 1 mM DTT, 75 mM NaCl, 0.05% BSA, 10 mg (mitochondria)or 1 mg (nuclear)extracts and 90 fmol of 32P-labeled double-strand uracil-containing oligonucleotide (U30)(Table 1)for 1 h at 321C. Reactions were terminated by addition of 1 ml each 5 mg/ml proteinase K and 10% SDS, and incubation for 15 min at 551C. To each tube, 20 ml formamide loading buffer (90% formamide, 10 mM EDTA, 1 mg/ml xylene cyanol FF and 1 mg/ml bromophenol blue)was added and the samples heated at 901C for 10 min. OGG1 activity was measured in a reaction (20 ml)containing 40 mM HEPES (pH 7.6), 5 mM EDTA, 1 mM DTT, 75 mM KCl, 10% glycerol, 100 mg (mitochondria)or 10 mg (nuclear) extracts and 88.7 fmol of 32P-labeled double-strand 8-oxoG- containing oligonucleotide (OG30)(Table 1)for 6 h at 321C.The reactions were terminated as described above. Endonuclease III homologue (NTH1)activity was determined in the same reaction conditions as for OGG1, with the exception that 50 mg (mitochondria)and 5 mg (nuclear)extracts were used, and the substrate was a 5-OHC-containing- oligonucleotide (OC30), incubated at 321C for 4 h. AP endonuclease activitiy was determined by incubating 1 mg mitochondrial protein with 100 fmol of a 32P-labeled tetrahydrofuran-containing duplexed 16-mer (Table 1)for 15 min at 321C, in 20 ml of reaction buffer containing 50 mM HEPES-KOH (pH 7.5), 50 mM KCl, 100 mg/ml BSA, 10 mM MgCl2, 10% glycerol and 0.05% Triton X-100. Reactions were terminated by the addition of 20 ml formamide loading buffer and heating at 901C for 10 min. For all incision assays, substrate and product DNAs were resolved by electrophoresis at 15 W for 1 h 10 min on 20% polyacrylamide gels containing 7 M urea. Bands were visualized by PhosphorImager and analysed using ImageQuantt (Molecular Dynamics). Incision activity was determined as the intensity of product bands relative to the total radioactivity in each lane.

Repair synthesis incorporation assay Repair synthesis reactions (50 ml)contained 40 m M HEPES (pH 7.6), 0.1 mM EDTA, 5 mM MgCl2, 0.2 mg/ml BSA, 50 mM KCl, 1 mM DTT, 25 mM phosphocreatine, 50 mg/ml Creatine Figure 7 Pol g activity stimulation by p53: increasing amounts of Phosphokinase, 2 mM ATP, 20 mM of each dATP, dTTP, mitochondrial extracts (0.25–5 mg)from wild-type (wt)or p53 dGTP and 2 mM of dCTP, 4 mCi a32PdCTP, 3% glycerol, 1 mM knockout (p53À/À)mice were incubated with 1 pmol of a 1 NAD þ , 300 ng of double-strand U-containing oligonucleo- nucleotide gap oligonucleotide, in the presence of 32P-dCTP, and gap-filing activity was quantified as the amount of radioactivity tide and the specified amounts of extracts. The reactions were incorporated, relative to 0.25 mg of MLME-wt (a). Alternatively, incubated at 321C for 2–4 h, with or without the addition of 5 mg of MLME from wt or p53À/À mice were incubated with 1 pmol 1 U of T4-DNA ligase. The DNA was extracted with equal of gap substrate alone or with increasing concentrations of volumes of phenol : chlorophorm : iso-amyl alcohol (25 : 24 : 1), recombinant p53 (b). Gap-filing activity was quantified as above, chlorophorm:iso-amyl alcohol (24 : 1)and precipitated by the relative to incorporation with 5 mg of MLME-wt in the absence of addition of 1/5 vol of 11 M NH3-acetate and 2.5 vol of cold added p53. Results presented are average7SEM of three and two ethanol. The DNA was dried and resuspended in formamide separate experiments loading dye and loaded onto a 20% acrylamide/7 M urea gel. The gels were resolved and visualized as described earlier. was concentrated in Centricon 10 micro-concentrators Gap-filling assay (Amicon)and the buffer exchanged to 20 m M HEPES (pH 7.6), 1 mM EDTA, 5 mM DTT, 20% glycerol, 100 mM Pol g activity was measured with a one-nucleotide gap filing KCl and protease inhibitors. Nuclear extracts were prepared assay, in a 10 ml reaction containing 40 mM HEPES (pH 7.6), from the pellets obtained after the first low-speed spin of 0.1 mM EDTA, 5 mM MgCl2, 0.2 mg/ml BSA, 50 mM KCl, the crude homogenates, using the same procedure described 1mM DTT, 25 mM phosphocreatine, 50 mg/ml Creatine Phos- above. All extracts were then aliquoted in small volumes phokinase, 2 mM ATP, 4 mM dCTP, 4 mCi a32PdCTP, 3% and kept frozen at À801C until use. Protein concentration glycerol, 1 mg poly dI Á dC, 1 pmol double-strand 1 nucleotide was determined using the Lowry method, with BSA as gap 34-mer oligonucleotide (GAP, Table 1)substrate and the standard. specified amounts of extracts. The reactions were incubated at

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6567 371C for 1 h and terminated by addition of 1 ml each 5 mg/ml monoclonal anti-lamin B2 (Novocastra); mouse monoclonal proteinase K and 10% SDS, and incubation for 15 min at anti-cytochrome oxidase IV (Molecular Probes); or goat 551C. The products were ethanol-precipitated, dried, sus- polyclonal anti-p53 (N-19)(Santa Cruz).For detection, pended in formamide loading dye and resolved in a 20% membranes were incubated with HRP-coupled secondary polyacrylamide/7 M urea gel, as previously described. antibodies (Santa Cruz)and the bands visualized using the ECL þ Pluss (Amersham-Pharmacia Biotech)kit. Electro-mobility shift assay (EMSA) Statistical analysis In total, 300 fmol of each oligonucleotide (Table 1)were incubated with 4 pmol of recombinant p53 in a 20 ml reaction The results are reported as mean7standard deviation of at containing: 25 mM HEPES pH 7.5, 15% glycerol, 50 mM NaCl, least three different mitochondrial and nuclear preparations, 1mM DTT and 10 mM EDTA. The reactions were incubated at each assayed twice. The differences among genotypes were 301C for 30 min and loaded onto a 5% polyacrylamide gel analysed by the Student’s t-test, and a Po0.05 was considered containing 5% glycerol. The gels were resolved at 200 V for statistically significant. 2h,at41C. The bands were visualized using PhosphoImager and the percentage of shifted oligonucleotides was calculated using the ImageQuant software. Abbreviations AP, apurinic/apyrimidinic; BER, base excision repair; MLME, Western blot analysis mouse liver mitochondrial extract; MLNE, mouse liver nuclear Proteins were separated on 12% Tris-glycine gels (Invitrogen); extract; mtBER, mitochondrial base excision repair; mtDNA, amounts of mitochondrial or nuclear extracts loaded are mitochondrial DNA; NTH1, endonuclease III homologue 1; specified in the figure legends. Transfer to PVDF membranes OGG1, oxoguanine DNA glycosylase; Pol g, DNA polymerase (Invitrogen)was carried out by electroblotting in transfer gamma; U, uracil; UDG, uracil DNA glycosylase. buffer (12 mM Tris, pH 8.3; 96 mM glycine, 20% methanol)for 2 h at 30 V. The membrane was blocked overnight at 41Cin Acknowledgements 5% nonfat dry milk (BioRad)in TBST (20 m M Tris-HCl pH We thank Drs Cayetano Von Kobbe and Syed Imam for the 7.2, 137 mM NaCl, 0.1 % Tween-20). Fresh milk-TBST was critical reading of this manuscript. We also thank Dr Draginja added with one of the following primary antibodies: mouse Djurickovic (NCIFCRF)for transferring the animals.

References

Allinson SL, Dianova II and Dianov GL. (2001). EMBO J., Longley MJ, Prasad R, Srivastava DK, Wilson SH and 20, 6919–6926. Copeland WC. (1998). Proc. Natl. Acad. Sci. USA, 95, Bohr VA, Stevnsner T and Souza-Pinto NC. (2002). Gene, 286, 12244–12248. 127–134. Marchenko ND, Zaika A and Moll UM. (2000). J. Biol. Degtyareva N, Subramanian D and Grifﬁth JD. (2001). Chem., 275, 16202–16212. J. Biol. Chem., 276, 8778–8784. Meira LB, Cheo DL, Hammer RE, Burns DK, Reis A and Domena JD and Mosbaugh DW. (1985). Biochemistry, 24, Friedberg EC. (1997). Nat. Genet., 17, 145. 7320–7328. Mihara M, Erster S, Zaika A, Petrenko O, Chittenden T, Dumont P, Leu JI, Della III PA, George DL and Murphy M. Pancoska P and Moll UM. (2003). Mol. Cell, 11, 577–590. (2003). Nat. Genet., 33, 357–365. Moll UM and Zaika A. (2001). FEBS Lett., 493, 65–69. Evans AR, Limp-Foster M and Kelley MR.. (2000). Mutat. Nilsen H, Otterlei M, Haug T, Solum K, Nagelhus TA, Res., 461, 83–108. Skorpen F and Krokan HE. (1997). Nucleic Acids Res., 21, Evans MK, Taffe BG, Harris CC and Bohr VA. (1993). 2579–2584. Cancer Res, 53, 5377–5381. Offer H, Milyavsky M, Erez N, Matas D, Zurer I, Harris CC Foord OS, Bhattacharya P, Reich Z and Rotter V. (1991). and Rotter V. (2001). Oncogene, 20, 581–589. Nucleic Acids Res., 19, 5191–5198. Offer H, Wolkowicz R, Matas D, Blumenstein S, Livneh Z and Gaiddon C, Moorthy NC and Prives C. (1999). EMBO J., 18, Rotter V. (1999). FEBS Lett., 450, 197–204. 5609–5621. Seo YR, Fishel ML, Amundson S, Kelley MR and Smith ML. Habano W, Sugai T, Nakamura SI, Uesugi N, Yoshida T and (2002). Oncogene, 21, 731–737. Sasou S. (2000). Gastroenterology, 118, 835–841. Sobol RW, Kartalou M, Almeida KH, Joyce DF, Engelward Hanawalt PC. (2002). Oncogene, 21, 8949–8956. BP, Horton JK, Prasad R, Samson LD and Wilson SH. Hofseth LJ, Khan MA, Ambrose M, Nikolayeva O, Welliver (2003). J. Biol. Chem., 278, 39951–39959. MX, Kartalou M, Hussain SP, Roth RB, Zhou X, Mechanic Souza-Pinto NC, Croteau DL, Hudson EK, Hansford RG and L, Zurer I, Rotter V, Samson LD and Harris CC. (2003). Bohr VA. (1999). Nucleic Acids Res., 27, 1935–1942. J. Clin. Invest., 112, 1887–1894. Souza-Pinto NC, Eide L, Hogue BA, Thybo T, Stevnsner T, Karahalil B, Souza-Pinto NC, Parsons JL, Elder RH and Bohr Seeberg E, Klungland A and Bohr VA. (2001). Cancer Res., VA. (2003). J. Biol. Chem., 278, 33701–33707. 61, 5378–5381. Krokan HE, Drablos F and Slupphaug G. (2002). Oncogene, Stierum RH, Dianov GL and Bohr VA. (1999a). Nucleic Acids 21, 8935–8948. Res., 27, 3712–3719. Krokan HE, Standal R and Slupphaug G. (1997). Biochem. J., Stierum RH, Dianov GL and Bohr VA. (1999b). Nucleic Acids 325, 1–16. Res., 27, 3712–3719. Lakshmipathy U and Campbell C. (1999). Mol. Cell. Biol., 19, Stuart JA, Karahalil B, Hogue BA, Souza-Pinto NC and Bohr 3869–3876. VA. (2004). FASEB J., 18, 595–597. Lee S, Elenbaas B, Levine A and Grifﬁth J. (1995). Cell, 81, Taguchi T, Ogihara M, Maekawa T, Hanaoka F and Tanno 1013–1020. M. (1995). Biochem. Biophys. Res. Commun., 216, 715–722.

Oncogene p53 in mtDNA repair NC de Souza-Pinto et al 6568 Vogelstein B, Lane D and Levine AJ. (2000). Nature, 408, Yamada NA and Farber RA. (2002). Cancer Res., 62, 307–310. 6061–6064. Wang XW, Yeh H, Schaeffer L, Roy R, Moncollin V, Egly Zhou J, Ahn J, Wilson SH and Prives C. (2001). EMBO J., 20, JM, Wang Z, Freidberg EC, Evans MK and Taffe BG. 914–923. (1995). Nat. Genet., 10, 188–195. Zurer I, Hofseth LJ, Cohen Y, Xu-Welliver M, Hussain Wilson III DM, Takeshita M, Grollman AP and Demple B. SP, Harris CC and Rotter V. (2004). Carcinogenesis, 25, (1995). J. Biol. Chem., 270, 16002–16007. 11–19.

Oncogene