We here reconstitute a minimal mammalian mitochondrial DNA (mtDNA) replisome in vitro. The mtDNA polymerase (POLc) cannot use double-stranded DNA (dsDNA) as template for DNA synthesis. Similarly, the TWINKLE DNA helicase is unable to unwind longer stretches of dsDNA. In combination, POLc and TWINKLE form a processive replication machinery, which can use dsDNA as template to synthesize single-stranded DNA (ssDNA) molecules of about 2 kb. The addition of the mitochondrial ssDNA-binding protein stimulates the reaction further, generating DNA products of about 16 kb, the size of the mammalian mtDNA molecule. The observed DNA synthesis rate is 180 base pairs (bp)/min, corresponding closely to the previously calculated value of 270 bp/min for in vivo DNA replication. Our findings provide the first biochemical evidence that TWINKLE is the helicase at the mitochondrial DNA replication fork. Furthermore, mutations in TWINKLE and POLc cause autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with deletions in mitochondrial DNA. The functional interactions between TWINKLE and POLc thus explain why mutations in these two proteins cause an identical syndrome.

Mutations in TWINKLE cause autosomal dominant progressive external ophthalmoplegia, a human disorder associated with multiple deletions in the mitochondrial DNA. TWINKLE displays primary sequence similarity to the phage T7 gene 4 primase-helicase, but no specific enzyme activity has been assigned to the protein. We have purified recombinant TWINKLE to near homogeneity and demonstrate here that TWINKLE is a DNA helicase with 5 to 3 directionality and distinct substrate requirements. The protein needs a stretch of 10 nucleotides of single-stranded DNA on the 5-side of the duplex to unwind duplex DNA. In addition, helicase activity is not observed unless a short single-stranded 3-tail is present. The helicase activity has an absolute requirement for hydrolysis of a nucleoside 5-triphosphate, with UTP being the optimal substrate. DNA unwinding by TWINKLE is specifically stimulated by the mitochondrial single-stranded DNA-binding protein.Our enzymatic characterization strongly supports the notion that TWINKLE is the helicase at the mitochondrial DNA replication fork and provides evidence for a close relationship of the DNA replication machinery in bacteriophages and mammalian mitochondria.The molecular mechanisms by which mtDNA is replicated in mammalian cells are of fundamental biological interest. Saccharomyces cerevisiae has served as a model system for studies of mammalian mtDNA replication, but there are significant differences between yeast and mammalian cells (1). Replication of the S. cerevisiae mtDNA is initiated from multiple sites of the ϳ86-kb genome, and the mtDNA molecules frequently undergo recombination. In contrast, the smaller mammalian mtDNAs (ϳ16 kb) initiate DNA replication from two specific origins of replication, oriH and oriL, and recombination is a rare or possibly even non-existent phenomenon (2).Mammalian mtDNA contains two major promoters, the light and heavy strand promoters, which produce near genomic length transcripts that, after RNA processing, release individual mRNAs, tRNAs, and rRNAs. A separate transcription unit for the rRNA genes in mammalian mitochondria has also been reported (3). Transcription from light strand promoters is not only necessary for gene expression but also produces the RNA primers required for initiation of mtDNA replication at oriH (1, 4). DNA synthesis from oriH is unidirectional and proceeds to displace the parental heavy strand. The nascent H strands frequently terminate 700 bp downstream of oriH, giving rise to 7 S DNA (D-loop strand). This termination event produces a characteristic triple-stranded structure, called the D-loop (5). The function of the D-loops is unknown, but they presumably play a role in regulating mtDNA replication.The mitochondrial DNA polymerase ␥ is a heterodimer comprising catalytic (A) and accessory (B) subunits of 140 and 54 kDa, respectively. The accessory subunit, polymerase ␥ B, which is not present in yeast, has been characterized as a processivity factor for the polymerase (6, 7). Polymerase ␥ B increases the affinity of the ...

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TWINKLE is a DNA helicase needed for mitochondrial DNA replication. In lower eukaryotes the protein also harbors a primase activity, which is lost from TWINKLE encoded by mammalian cells. Mutations in TWINKLE underlie autosomal dominant progressive external ophthalmoplegia (adPEO), a disorder associated with multiple deletions in the mtDNA. Four different adPEO-causing mutations (W315L, K319T, R334Q, and P335L) are located in the N-terminal domain of TWINKLE. The mutations cause a dramatic decrease in ATPase activity, which is partially overcome in the presence of single-stranded DNA. The mutated proteins have defects in DNA helicase activity and cannot support normal levels of DNA replication. To explain the phenotypes, we use a molecular model of TWINKLE based on sequence similarities with the phage T7 gene 4 protein. The four adPEO-causing mutations are located in a region required to bind single-stranded DNA. These mutations may therefore impair an essential element of the catalytic cycle in hexameric helicases, i.e. the interplay between single-stranded DNA binding and ATP hydrolysis.