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. 2016 May 19:6:26414.
doi: 10.1038/srep26414.

Irc3 is a mitochondrial DNA branch migration enzyme

Ilja Gaidutšik  1 , Tiina Sedman  1 , Sirelin Sillamaa  1 , Juhan Sedman  1
Affiliations

Irc3 is a mitochondrial DNA branch migration enzyme

Ilja Gaidutšik et al. Sci Rep. .

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.

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Figures

Figure 1
Figure 1. Irc3 modulates the formation of branched intermediates of mitochondrial DNA.
(a) A map of the 1.8 kb a11 rho− mitochondrial DNA (mtDNA) repeat containing actively transcribed ori3 with characteristic A, B and C boxes. The coordinates correspond to the reference yeast mtDNA sequence (Genbank: KP263414.1). (b) A scheme of 2D-gel electrophoresis analysis. ds: linear double-stranded molecules; x: X-arc; y: Y-arc; 1N: unit size (1.8 kb) molecules; 2N: double-unit size (3.6 kb) molecules, IR: irregular branched DNA molecules. (c,d) 2D analysis of mtDNA isolated from log phase cultures of the a11 strain with the indicated nuclear mutations. mtDNA was digested with XbaI (c) or with S1 and XbaI (d) and separated by 2-D agarose gels followed by blot hybridization to the a11 repeat probe.
Figure 2
Figure 2. Irc3 binds preferentially to branched-DNA.
(a,b) Electrophoresis mobility shift assays (EMSA) using indicated concentrations of Irc3 and 32P-labeled probes mimicking the structure of a replication fork (a) or X-shaped and linear DNA (b), as schematically depicted below the panels. Position of the 5′ label in the DNA substrates is indicated by a star. DSF: fork with both arms double-stranded; LDF- fork with missing nascent lagging strand; LGF- fork with missing nascent leading fork, SSF:fork with both arms single-stranded and X0: immovable four-way junction; X12- four-way junction containing a movable core flanked by heterologous sequences of 19 or 20 bp in each arm; 49DS: 49 bp dsDNA; 49SS: 49 nt ssDNA. (c) Stimulation of Irc3 ATPase activity with different DNA cofactors described for panels a,b. All assays were performed as triplicates using 15 nM Irc3 and the error bars indicate SD. The significance of differences between Irc3 ATPase activities stimulated by the linear 49 bp dsDNA and various branched-DNA cofactors was determined using Student’s t-test for unpaired observations. *P < 0.05; **P < 0.01; ***P < 0.001.
Figure 3
Figure 3. Irc3 unwinds stalled replication forks.
(a,b) Irc3 is inactive on linear DNA substrate molecules with a 5′ (a) and 3′ (b) single-stranded extension or with a single-stranded fork structure (c). (d–f) Irc3 unwinds the lagging- or leading-strand analog from a branched DNA substrates that mimic defective replication forks. LDF- fork missing nascent lagging strand (d,f); LGF- fork missing nascent leading fork (e,f). Irc3 was 5 nM in all reactions.
Figure 4
Figure 4. Irc3 preferentially reverses replication fork structures and possesses branch-migrating activity.
Comparison of Irc3 dependent unwinding of model fork substrates with homologous (HOM) (a,e) or heterologous (HET) arms (b,e). (c,d,f) Irc3 dependent unwinding of four way junctions with fixed immobile structure (c,f) or junctions with a mobile 12 nt core (d,f). Irc3 was 5 nM in all reactions.
Figure 5
Figure 5. RecG complements partially the loss of Irc3 in yeast mitochondria.
(a) Graphic representation of RecG and Irc3 sequences. MTS- cleavable mitochondrial targeting sequence; Helicase I and Helicase II- helicase domains; Wedge- RecG wedge domain; CTE- C-terminal extension of Irc3. (b) Growth curves of yeast cells in SC –Leu medium containing 3% glycerol as a carbon source. Δirc3 + IRC3- red squares; Δirc3 + RecG- green triangles; Δirc3 + control plasmid- black circles. (c–e) Loss of respiratory competence of yeast cells during growth on glucose, as revealed by plating out equal number of cultivated cells onto glycerol and glucose containing agar plates. In d and e, Δirc3 + RecG and Δirc3 strain viability is shown using different scale of the y axis. Three biological replicas were analyzed and the error bars represent minimum and maximum values of the measurements. (f) Mitochondrial DNA fragments in Δirc3 strain of W303-1B background. Mitochondrial DNA was isolated from the indicated strains, digested with EcoRV prior electrophoresis and blot-detection for a 1.8 kb cox2 fragment. Different samples were normalized to equalize the signals of the 1.8 kb fragments. (g) Quantification of DNA fragments in the range of 0.2–0.5 kb and 0.5–0.7 kb in the RV digested mtDNA of the indicated strains. Three biological replicas were analyzed and the error bars indicate SD.
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