Mitochondrial localization of p53 was observed in stressed and unstressed cells. p53 is involved in DNA repair and apoptosis. It exerts physical and functional interactions with mitochondrial DNA and DNA polymerase c (pol c). The functional cooperation of p53 and pol c during DNA synthesis was examined in the mitochondrial fraction of p53-null H1299 cells, as the source of pol c. The results show that p53 may affect the accuracy of DNA synthesis in mitochondria: (1) the excision of a misincorporated nucleotide increases in the presence of (a) recombinant wild-type p53 (wtp53); (b) cytoplasmic fraction of LCC2 cells expressing endogenous wtp53 (but not specifically pre-depleted fraction); (c) cytoplasmic extract of H1299 cells overexpressing wtp53, but not exonuclease-deficient mutant p53-R175H. (2) Mitochondrial extracts of HCT116(p53 þ / þ ) cells display higher exonuclease activity compared with that of HCT116(p53À/À) cells. Addition of exogenous p53 complements the HCT116(p53À/À) mitochondrial extract mispair excision. Furthermore, the misincorporation was lower in the mitochondrial fraction of HCT116(p53 þ / þ ) cells as compared with that of HCT116(p53À/À) cells. The tumor suppressor protein p53 represents a central factor for the maintenance of genome stability and for the suppression of cancer. 1 p53 is present at low levels in unstressed cells, but after exposure to various stress signals, the protein is stabilized and activated by a series of post-translational modifications. p53 is involved in the induction of cell-cycle arrest and apoptosis through transcriptional activation of target genes. 2 p53 displays multicompartmental functions in the cell. Interestingly, in response to multiple death stimuli, mitochondrial p53 targeting occurs in a wide spectrum of cell types, where it elicits a series of responses that can promote an apoptosis through a transcription-independent mechanism. 3,4 Several lines of evidence support a connection between p53 and mitochondrion. Mitochondrial p53 levels are proportional to total p53 levels, and a small fraction of protein is associated with mitochondrial DNA (mtDNA) in the absence of exogenous stress, thus implying a non-apoptotic function for mitochondrial p53. 5 Furthermore, translocation of p53 to mitochondria was observed in various cells (e.g., MCF-7 and HCT116) independent of apoptosis. 6 p53 may be a component of a stress response pathway that involves the upregulation of mitochondrial repair in mouse liver and cancer cells, suggesting the potential role of p53 in maintaining mtDNA stability. 7,8 Mitochondrial DNA, a 16.5-kb circular double-stranded molecule, is replicated by an assembly of enzymes and proteins consisting of DNA polymerase g (pol g), ssDNAbinding protein, DNA helicase and various accessory proteins and transcription factors. 9 Mitochondrial DNA is prone to mutations, as it is localized near the inner mitochondrial membrane in which reactive oxygen species are generated. In addition, mtDNA lacks histone protection and highly efficient DNA repair...

p53 in mitochondria may functionally interact with DNA polymerase gamma, thus providing a proofreading function during mitochondrial DNA replication for excision of nucleoside analogs and polymerization errors.

p53 in cytoplasm displays an intrinsic 3'-->5' exonuclease activity. To understand the significance of p53 exonuclease activity in cytoplasm, cytoplasmic extracts of various cell lines were examined for exonuclease activity with different single-stranded RNA (ssRNA) substrates. Using an in vitro RNA degradation assay, we observed in cytoplasmic extracts of LCC2 cells, expressing high levels of endogenous wtp53, an efficient 3'-->5' exonuclease activity with RNA substrates, removing the 3'-terminal nucleotides. Interestingly, RNA containing AU-rich sequences (ARE) is the permissive substrate for exonucleolytic degradation. Evidence that exonuclease function with RNA detected in cytoplasmic extracts is attributed to the p53 is supported by several facts: (1) this activity closely parallels with status and levels of endogenous cytoplasmic p53; (2) the endogenous exonuclease exerts identical RNA substrate specificity and excision profile characteristic for purified baculovirus-or bacterially-expressed wtp53s; (3) the exonuclease activity with ARE RNA is competed out by the presence of ss or double-stranded DNA substrate utilized by p53 protein in cytoplasm; (4) immunoprecipitation by specific anti-p53 antibodies markedly reduced the exonuclease activity with both RNA and DNA substrates; and (5) transfection of the wtp53, but not exonuclease-deficient mutant p53-R175H, into p53-null H1299 or HCT116 cells induced high levels of exonuclease activity with ARE RNA substrate in cytoplasm with characteristic excision profile. The efficient ARE RNA degradation correlates with the efficient binding of p53 to ARE RNA in cytoplasm. The possible role of p53 exonuclease activity in ARE-mRNA destabilization in cytoplasm, which may be important for expression of proteins that control cell growth and/or apoptosis is discussed.

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