Human mitochondrial helicase Twinkle has RNA binding, annealing, and strand-exchange activities.

Twinkle is the sole replicative helicase in human mitochondria, essential for mitochondrial DNA replication. Beyond its canonical unwinding activity, Twinkle has non-canonical activities, including DNA annealing and strand-exchange. Here, we show that these non-canonical activities extend to RNA. Twinkle binds RNA and catalyzes RNA:DNA hybrid formation through annealing, strand-exchange, and toehold-mediated strand displacement. Twinkle can unwind RNA:DNA forks when loaded onto the DNA tail but not the RNA tail. Although the physiological role of these RNA-related activities remains unclear, we show that Twinkle can strand-exchange an RNA downstream of a stalled replication fork to restart replication. The annealing/strand-exchange activity can be involved in DNA replication initiation and repair, but RNA:DNA hybrids can compromise genome integrity, emphasizing the need to balance unwinding and annealing activities. Interestingly, mitochondrial SSB inhibits the RNA:DNA annealing activity of Twinkle, thus regulating the non-canonical functions of Twinkle. A disease-associated W315L variant, which is defective in DNA replication, retains annealing and strand-exchange functions with both RNA and DNA, resulting in an imbalance between replication and annealing functions that may underlie its pathogenicity. Our findings of Twinkle's RNA-binding and strand-exchange activities may have a connection to its localization within mitochondrial RNA granules.