TWINKLE Has 5' -> 3' DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein.
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.
Korhonen
Jenny A
JA
Department of Medical Nutrition, Karolinska Institutet, Novum, SE-141 86 Huddinge, Sweden.
Gaspari
Martina
M
Falkenberg
Maria
M
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D019915:DNA Primase; D004265:DNA Helicases