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

Summary

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 (Irc3_op) that throughout its helicase motor domain has approximately 82% similarity with the S. cerevisiae protein. Irc3_op retains enzymatic activity at considerably higher temperatures than Irc3_sc, displaying the fastest rate of ATP hydrolysis at 41 °C and a Tm of 45.3 °C for its secondary structure melting. We demonstrate that Irc3_op is a structure-specific DNA helicase translocating on both single- and double-stranded DNA molecules. Like the homolog of S. cerevisiae, Irc3_op can unwind only DNA molecules that contain branched structures. Different DNA molecules containing three- and four-way branches are utilized by Irc3_op as unwinding substrates. Importantly, the preferred unwinding substrate of Irc3_op 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 Irc3_op only partially complements the irc3Δ phenotype in S. cerevisiae.