Irc3 is a monomeric DNA branch point‐binding helicase in mitochondria of the yeast Saccharomyces cerevisiae

Irc3 is a superfamily II DNA helicase required for the maintenance of mitochondrial DNA stability in Saccharomyces cerevisiae . Here, we show that recombinant Irc3 is a monomeric protein and that it can form a binary complex with forked DNA. The catalytically active enzyme is a monomer as no positive cooperativity of ATP hydrolysis or DNA unwinding can be detected. Interestingly, we find that Irc3 prefers to unwind the nascent lagging strand at a replication fork. Using DNase I footprinting, we demonstrate that Irc3 captures DNA substrates by establishing a strong contact at the DNA branching point. Additional protections on the lagging strand template suggest a 3′‐to‐5′ polarity for Irc3 movement.