Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance

Abstract

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability.

Irc3 is a mitochondrial DNA branch migration enzyme

“…1c , irc3Δ). This result was similar to that previously observed with a different rho − strain a1184 23 . Furthermore, in the S1 treated a11 irc3 Δ mtDNA samples, we could also hardly visualize the regular Y and X structures ( Fig.…”

“…In accordance with our previous analysis, Irc3 did not unwind different linear DNA substrates with either 5′ or 3′ single-stranded extensions of 15–25 nt or with a 25 nt single-stranded fork structure at the end of a short double-stranded region ( Fig. 3a–c 23 ). However, preferential complex formation with branched DNA molecules and strong stimulation of Irc3 ATPase by the same cofactors suggested that Irc3 could unwind substrates resembling four-way Holliday junctions or replication forks.…”

“…As previously reported, the ssDNA cofactor did not stimulate Irc3 ATPase ( Fig. 2c and 23 ). Preferential binding of Irc3 to Y and X-shaped DNA and strong stimulation of Irc3 ATPase activity with branched DNA cofactors suggested that Irc3 could function as branched DNA specific enzyme.…”

“…We have previously reported that Irc3 is directly involved in mtDNA metabolism in both rho + and rho − yeast strains 23 . Irc3 is a member of the Superfamily II of helicases and ATP-dependent DNA translocases and irc3 Δ yeast strains are characterized by excessive fragmentation of mtDNA.…”

“…Our recent study revealed that Irc3 - a dsDNA dependent ATPase of the Superfamily II- is essential for yeast mtDNA maintenance 23 . irc3 Δ mutant yeast cells accumulated double-stranded breaks in mtDNA and lose the wild-type mitochondrial genome in the course of a few generations of growth on glucose-containing media.…”