In Vitro Assays of TWINKLE Function

TWINKLE is an essential helicase that unwinds the duplex mitochondrial genome during DNA replication. In vitro assays using purified recombinant forms of the protein have been an instrumental tool for gaining mechanistic insights about TWINKLE and its function at the replication fork. Here we present methods to probe the helicase and ATPase activities of TWINKLE. For the helicase assay, TWINKLE is incubated with a radiolabeled oligonucleotide annealed to an M13mp18 single-stranded DNA template. TWINKLE will displace the oligonucleotide, which is then visualized by gel electrophoresis and autoradiography. To measure the ATPase activity of TWINKLE, a colorimetric assay is used, which quantifies the release of phosphate upon ATP hydrolysis by TWINKLE.