Abstract
Mutations in mitochondrial DNA is an outcome of errors produced by DNA polymerase γ during replication and failure of the repair mechanism. Misincorporation of non-canonical dUTP leads to mutagenesis or apoptosis, and may contribute to the cytotoxic effects of 5′-fluorouracil chemotherapy. Tumor suppressor p53 protein in the mitochondria displays physical and functional interactions with mitochondrial DNA and polymerase γ, and by its intrinsic 3′→5′ exonuclease activity can diminish the polymerization errors. Here we demonstrate the impact of p53 on incorporation of uracil into DNA examined with mitochondrial fractions, as the source of polymerase γ. p53 in mitochondria facilitates DNA damage repair functions resulting from uracil–DNA misincorporation. Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, thus expanding the spectrum of DNA damage sites exploited for proofreading as a trans-acting protein. The data suggest that p53 may contribute to defense of the cells from consequences of dUTP misincorporation in both normal and tumor cells.
Highlights
A high frequency of mutations within mitochondrial DNA, resulting in mitochondrial dysfunctions, are an important source of various diseases including cancer and human aging [1, 2]
Our biochemical studies revealed that the procession of U:A and mismatched U:G lesions enhances in the presence of recombinant or endogenous cytoplasmic p53. p53 in mitochondria can function as an exonuclease/proofreader for polymerase γ by either decreasing the incorporation of non-canonical dUTP into DNA or by promoting the excision of incorporated nucleotide from nascent DNA, expanding the spectrum of DNA damage sites exploited for proofreading as a transacting protein
The functional interaction between p53 and pol g during incorporation of dU was assessed during DNA synthesis catalyzed by the replication machinery of mitochondrial fractions derived from isogenic HCT116 cells, as a model system [22]
Summary
A high frequency of mutations within mitochondrial DNA (mtDNA), resulting in mitochondrial dysfunctions, are an important source of various diseases including cancer and human aging [1, 2]. MtDNA integrity and replication rest on numerous nucleus-encoded DNA repair proteins normally localized in mitochondrial matrix or imported upon various stress signals [8]. In addition to regulation of apoptosis by p53 via proteinprotein interaction at the mitochondrial outer membrane, the biological routes regulated by p53 comprises mtDNA synthesis/repair for mitochondrial function after translocating to matrix [14]. Co-localization of p53 with mtDNA, pol g and various constituents of the mtDNA replication apparatus (e.g. transcription factor A-TFAM and single-stranded DNA-binding proteins), even in the absence of exogenous stress independent of apoptosis, establishes a non-apoptotic function for matrix-localized p53 which underlines an importance of p53 in mtDNA homeostasis [18,19,20]. B)Intra-mitochondrial p53 provides an error-repair proofreading function for pol γ by excision of misincorporated nucleotides [22]. The functional interaction of p53 and pol γ is significant for avoiding mtDNA mutations and mtDNA depletions that are frequently observed in human cancers and neurodegenerative diseases [13]
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