Mitochondrial helicase Irc3 translocates along double-stranded DNA

Irc3 is a superfamily II helicase required for mitochondrial DNA stability in Saccharomyces cerevisiae. Irc3 remodels branched DNA structures, including substrates without extensive single-stranded regions. Therefore, it is unlikely that Irc3 uses the conventional single-stranded DNA translocase mechanism utilized by most helicases. Here, we demonstrate that Irc3 disrupts partially triple-stranded DNA structures in an ATP-dependent manner. Our kinetic experiments indicate that the rate of ATP hydrolysis by Irc3 is dependent on the length of the double-stranded DNA cosubstrate. Furthermore, the previously uncharacterized C-terminal region of Irc3 is essential for these two characteristic features and forms a high affinity complex with branched DNA. Together, our experiments demonstrate that Irc3 has double-stranded DNA translocase activity.