UvrD-like helicase Hmi1 Has an ATP independent role in yeast mitochondrial DNA maintenance.

Hmi1 is a UvrD-like DNA helicase required for the maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Deletion of the HMI1 ORF leads to the formation of respiration-deficient petite mutants, which either contain a short fragment of mtDNA arranged in tandem repeats or lack mtDNA completely. Here we characterize point mutants of the helicase designed to target the ATPase or ssDNA binding activity and show that these mutations do not separately lead to complete loss of the Hmi1 function. The mutant strains support ATP production via oxidative phosphorylation and enable us to directly analyze the impact of both activities on the stability of wild-type mtDNA in this petite-positive yeast. Our data reveal that Hmi1 mutants affecting ssDNA binding display a stronger defect in the maintenance of mtDNA compared to the mutants of ATP binding/hydrolysis. Hmi1 mutants impaired in ssDNA binding demonstrate sensitivity to UV irradiation and lower levels of Cox2 encoded by the mitochondrial genome. This suggests a complex and multifarious role for Hmi1 in mtDNA maintenance-linked transactions, some of which do not require the ATP-dependent helicase activity.

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 polymorpha that retains structural integrity and catalytic activity at temperatures above 40 °C. Irc3op can complement the respiratory deficiency phenotype of a S. cerevisiae irc3Δ 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.

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.