Background: TWINKLE is the human mitochondrial DNA helicase associated with heritable neuromuscular diseases. Results: TWINKLE has NTPase-dependent DNA unwinding activity and NTPase-independent DNA annealing activity. The unwinding activity is enhanced by displaced strand traps. Conclusion: TWINKLE has more than one ssDNA-binding sites, the one associated with annealing interferes with unwinding in the absence of traps. Significance: The annealing activity may be involved in recombination-mediated replication initiation.

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.

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TWINKLE is the only known helicase at the human mitochondrial DNA (HmtDNA) replication fork. It is encoded by PEO1 gene of chromosome 10. It shares 46% amino acid sequence similarity with the helicase T7gene4(gp4) from bacteriophage. Mutations in PEO1 have been linked to deletions in the mitochondrial DNA causing neuromuscular disorders in human. To understand the diseases better, it is crucial to define the molecular mechanism of HmtDNA replication guided by the proteins involved in it. In this study, we have focused on understanding the kinetics of dsDNA unwinding by TWINKLE using ensemble assay with radiolabeled DNA substrates. We have observed that the characteristic unwinding activity of TWINKLE is markedly different from T7gp4. Our most interesting finding indicates that the morphology of non‐translocating strand plays a major role during unwinding by TWINKLE. This is an important step towards understanding the mechanism of initiation of replication from the D‐loop in the mitochondrial DNA. Our final goal is to understand the mechanism of replication of mitochondrial DNA in vitro which in turn would allow us to define the molecular basis of the diseases in human.