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

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P53 Functions in the Incorporation Step in DNA Base Excision Repair in Mouse Liver Mitochondria 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 statisti- cally 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.
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