Irc3 is a mitochondrial DNA branch migration enzyme

Abstract

Integrity of mitochondrial DNA (mtDNA) is essential for cellular energy metabolism. In the budding yeast Saccharomyces cerevisiae, a large number of nuclear genes influence the stability of mitochondrial genome; however, most corresponding gene products act indirectly and the actual molecular mechanisms of mtDNA inheritance remain poorly characterized. Recently, we found that a Superfamily II helicase Irc3 is required for the maintenance of mitochondrial genome integrity. Here we show that Irc3 is a mitochondrial DNA branch migration enzyme. Irc3 modulates mtDNA metabolic intermediates by preferential binding and unwinding Holliday junctions and replication fork structures. Furthermore, we demonstrate that the loss of Irc3 can be complemented with mitochondrially targeted RecG of Escherichia coli. We suggest that Irc3 could support the stability of mtDNA by stimulating fork regression and branch migration or by inhibiting the formation of irregular branched molecules.

Mitochondrial helicase Irc3 translocates along double‐stranded DNA

“…We also find that the C terminus of Irc3 is responsible for high affinity binding of branched DNA. These data together with our previous study , indicate that Irc3 utilizes the dsDNA translocase activity to remodel branched DNA intermediates. Similar biochemical properties have been described for the T4 UvsW and bacterial RecG helicase, the yeast remodeler Rad5, and the related Swi/Snf2 family proteins SMARCAL1 and HTLF in higher eukaryotes .…”

“…Irc3 and Irc3‐K65A correspond to the mature mitochondrial polypeptide (aminoacid residues 29–689) and were purified as previously described with the following modifications: 0.1% Tween 20 was included in purification buffers; the cleared lysate was prefractionated by 30–60% (NH 4 ) 2 SO 4 ; TEV protease was used at 0.1 mg/1 mg protein for 6–8 h at 4 °C; S‐Sepharose fractions containing Irc3 were purified on Heparin‐FF (GE Healthcare). The purification scheme of Irc3 was followed for Irc3‐∆C(29–482) and Irc3‐∆N(386–689). Purified proteins (Fig.…”

“…Recently, we demonstrated that Irc3, a previously uncharacterized fourth member of the SFII, is a DNA helicase . Irc3 demonstrates some sequence similarity to Class I restriction endonucleases, but the homology is limited to the motor domains and therefore Irc3 appears to represent a separate subfamily of the SFII helicases . Irc3 is required for the stability of yeast mitochondrial DNA (mtDNA) as ∆ irc3 strains display increased levels of dsDNA breaks, rapidly lose their wt mitochondrial genome and accumulate aberrant rho − mtDNA variants .…”

“…Irc3 is required for the stability of yeast mitochondrial DNA (mtDNA) as ∆ irc3 strains display increased levels of dsDNA breaks, rapidly lose their wt mitochondrial genome and accumulate aberrant rho − mtDNA variants . In vitro experiments have demonstrated that Irc3 has branched DNA‐specific helicase and branch migration activity suggesting involvement in recombination‐related processes, such as replication fork regression or inhibition of illegitimate recombination . Importantly, the yeast ∆irc3 phenotype can be partially complemented with RecG of Escherichia coli targeted to mitochondria, suggesting that RecG and Irc3 could have similar biochemical functions .…”

RecG controls DNA amplification at double‐strand breaks and arrested replication forks

“…However, recently several candidates have been proposed. These include the mitochondrial helicase Irc3 of Saccharomyces cerevisiae , the plastid and mitochondrial helicase RECG of Physcomitrella patens , the mitochondrial helicase RECG1 of Arabidopsis thaliana and the human nuclear helicase SMARCAL1 . All of these genes are implicated in the maintenance of DNA stability and all the plastid and mitochondrial genes show partial cross‐complementation with recG .…”