TWINKLE Has 5′ → 3′ DNA Helicase Activity and Is Specifically Stimulated by Mitochondrial Single-stranded DNA-binding Protein

Abstract

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 ...

Mitochondrial pyrimidine nucleotide carrier (PNC1) regulates mitochondrial biogenesis and the invasive phenotype of cancer cells

“…As can be seen in Figure 3d, relative mtDNA transcription per mitochondrion was decreased in cells with suppressed PNC1, whereas it was increased in cells with overexpressed PNC1. UTP is a cofactor for the DNA helicase twinkle, which is necessary for mtDNA replication (Korhonen et al, 2003). Thus, PNC1 expression would also be expected to alter mtDNA replication.…”

“…C Favre et al although twinkle can use different 5 0 -triphosphates as cofactors for its helicase activity, UTP is proposed to be its most potent cofactor (Korhonen et al, 2003). Because PNC1 imports UTP into mitochondria, it is rational to propose that decreased mitochondrial UTP in PNC1-deficient cells (Floyd et al, 2007) would reduce the efficiency of twinkle activity and reduce mtDNA levels.…”

Unraveling the structure of DNA during overstretching by using multicolor, single-molecule fluorescence imaging

“…The PCR fragment was cloned into pBacPAK9 and used to generate recombinant Autographa californica nuclear polyhedrosis virus (BacPAK, Clontech). The protein was expressed in Spodoptera frugiperda (Sf9) cells and purified as previously described (32), with an additional purification step (before the hydroxyapatite column) using a 1-ml HiTrap SP column (GE Healthcare). The purified mtSSB cysteine variant was stored in 10 mM KPO4 pH 7.2, 0.1 M NaCl and 10% glycerol, and labeled with maleimide Alexa-555 dye (Molecular Probes).…”

Human mitochondrial RNA polymerase primes lagging-strand DNA synthesis in vitro

“…TWINKLE, mtSSB, POL␥A, POL␥B, and TFAM were expressed and purified as described (22,32). To generate POLRMT we coinfected insect cells with viruses expressing both POLRMT and TFB2M and purified the heterodimeric complexes to near homogeneity over Ni 2ϩ -agarose, as described in ref.…”