Irc3 of the thermotolerant yeast Ogataea polymorpha is a branched-DNA-specific mitochondrial helicase

The Saccharomyces cerevisiae Irc3 protein is a mitochondrial Superfamily II DNA helicase that, according to genomic data, is conserved in different yeast species. Here we characterize Irc3 helicase from the thermotolerant yeast Ogataea polymorpha (Irc3op) that throughout its helicase motor domain has approximately 82% similarity with the S. cerevisiae protein. Irc3op retains enzymatic activity at considerably higher temperatures than Irc3sc, displaying the fastest rate of ATP hydrolysis at 41 ÅC and a Tm of Å45.3 C for its secondary structure melting. We demonstrate that Irc3op is a structure-specific DNA helicase translocating on both single- and double-stranded DNA molecules. Like the homolog of S. cerevisiae , Irc3op can unwind only DNA molecules that contain branched structures. Different DNA molecules containing three- and four-way branches are utilized by Irc3op as unwinding substrates. Importantly, the preferred unwinding substrate of Irc3op is a DNA fork containing the nascent lagging strand, suggesting a possible role in replication restart following a block in leading strand polymerization. A lower unwinding efficiency of four-way branched DNA molecules could explain why Irc3op only partially complements the irc3delta phenotype in S. cerevisiae . ### Competing Interest Statement The authors have declared no competing interest.