Mitochondrial helicase involved in mtDNA replication and repair (By similarity). Might have a role in mtDNA repair (By similarity). Has DNA strand separation activity needed to form a processive replication fork for leading strand synthesis which is catalyzed by the formation of a replisome complex with POLG and mtSDB (By similarity). Preferentially unwinds DNA substrates with pre-existing 5'-and 3'- single-stranded tails but is also active on a 5'- flap substrate (By similarity). Can dissociate the invading strand of immobile or mobile D-loop DNA structures irrespective of the single strand polarity of the third strand (By similarity). In addition to its DNA strand separation activity, also has DNA strand annealing, DNA strand-exchange and DNA branch migration activities (By similarity). ATP + H2O = ADP + H(+) + phosphate Homohexamer (via C-terminus) which assembled in a ring-like structure. Homoheptamer which assembled in a ring-like structure. Oligomerization is Mg(2+), nucleotide and DNA-independent, however, Mg(2+) and nucleotide stabilize the homohexameric form. Interacts with POLG in vitro. Interacts with LONP1. Colocalizes with mtDNA in mitochondrial nucleoids, a nucleoproteins complex consisting of a number of copies of proteins associated with mtDNA, probably involved in mtDNA maintenance and expression. Ubiquitous with the highest levels in the liver, heart and kidneys. The skeletal muscle, brain and testis showed lower but detectable expression. Expression is coregulated with MRPL43. N-terminus enhances protein stability and hexamer formation, which is important for DNA binding, and is required for DNA helicase activity and, ultimately, for mtDNA replisome processivity. The N-terminus contains a putative primase-like domain; however the absence of the zinc binding domain and other motifs important for catalysis suggests that TWNK lacks primase activity.