Human Mitochondrial DNA Helicase TWINKLE Is Both an Unwinding and Annealing Helicase

Abstract

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.

Biochemical Characterization of the Human Mitochondrial Replicative Twinkle Helicase

“…When ATP is added, ϳ17 and 8% of the molecules display clear unwinding/reannealing and unwinding events, respectively. These results are consistent with a previous bulk study that showed Twinkle annealing activity can take place in the presence of a non-hydrolyzable nucleotide analogue or in the absence of nucleotide, but rates of annealing increased when a hydrolyzable nucleotide was added (12).…”

“…Here we observed that Twinkle unwound the 5Ј tailed duplex substrate (substrate 5) at ϳ5-fold greater rate than the 3Ј tailed duplex (substrate 6) (Fig. 3B), consistent with its preferential 5Ј to 3Ј directionality of unwinding under multi-turnover conditions (10,12).…”

“…Notably, Twinkle unwound the downstream 5Ј flap oligonucleotide of the 5Ј flap DNA substrate (Fig. 1B), consistent with its 5Ј to 3Ј directionality of unwinding (10,12). Conversely, Twinkle showed relatively poor activity on a 3Ј flap substrate (substrate 3) that lacks a pre-existing 5Ј single-stranded tail (Fig.…”

“…Twinkle catalyzes unwinding of duplex DNA with a 5Ј to 3Ј directionality fueled by the hydrolysis of nucleoside triphosphate (10 -12). Twinkle can load onto single-stranded DNA circles (10,12) or doublestranded DNA bubble structures (10), and it binds both singlestranded and double-stranded DNA (12). Twinkle anneals complementary single-stranded DNA (12), but the physiological significance of this biochemical activity remains to be understood.…”

“…Twinkle can load onto single-stranded DNA circles (10,12) or doublestranded DNA bubble structures (10), and it binds both singlestranded and double-stranded DNA (12). Twinkle anneals complementary single-stranded DNA (12), but the physiological significance of this biochemical activity remains to be understood. Although Twinkle plays a key role in separating strands at the mitochondrial replication fork, the enzyme is weakly active on G-quadruplex DNA structures (13), which are likely to form in the guanine-rich strand of the mitochondrial genome (13,14).…”