Mitochondrial Irc3 helicase of the thermotolerant yeast Ogataea polymorpha displays dual DNA- and RNA-stimulated ATPase activity

Irc3 is one of the six mitochondrial helicases described in Saccharomyces cerevisiae. Physiological functions of Irc3 are not completely understood as both DNA metabolic processes and mRNA translation have been suggested to be direct targets of the helicase. In vitro analysis of Irc3 has been hampered by the modest thermostability of the S. cerevisiae protein. Here, we purified a homologous helicase (Irc3op) of the thermotolerant yeast Ogataea pol-ymorpha that retains structural integrity and catalytic activity at temperatures above 40 degrees C. Irc3op can com-plement the respiratory deficiency phenotype of a S. cerevisiae irc3 Delta mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op is best stimulated by branched and double-stranded DNA cofactors. Single-stranded DNA binds Irc3op tightly but is a weak activator of the ATPase activity. We could also detect a lower level stimulation with RNA, especially with molecules possessing a compact three-dimensional structure. These results support the idea that that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo. Furthermore, our analysis of kinetic parameters predicts that Irc3 could have a regulatory function via sensing changes of the mitochondrial ATP pool or respond to the accumulation of single-stranded DNA.

Automated summary

Irc3 is a mitochondrial helicase in Saccharomyces cerevisiae that has been suggested to play a role in both DNA metabolic processes and mRNA translation. However, the modest thermostability of the S. cerevisiae protein has limited in vitro analysis. In this study, a homologous helicase from the thermotolerant yeast Ogataea polymorpha (Irc3op) was purified, which retained its structural integrity and catalytic activity at temperatures above 40 degrees C. Irc3op could complement the respiratory deficiency phenotype of a S. cerevisiae irc3 Delta mutant, indicating conservation of biochemical functions. The ATPase activity of Irc3op was best stimulated by branched and double-stranded DNA cofactors, and had lower but detectable stimulation with RNA molecules possessing a compact three-dimensional structure. These findings suggest that Irc3 might have dual specificity and remodel both DNA and RNA molecules in vivo.