Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation

Abstract

Mitochondria contain tens to thousands of copies of their own genome (mitochondrial DNA [mtDNA]), creating genetic redundancy capable of buffering mutations in mitochondrial genes essential for cellular function. However, the mechanisms regulating mtDNA copy number have been elusive. Here we found that DNA synthesis and degradation by mtDNA polymerase γ (POLG) dynamically controlled mtDNA copy number in starving yeast cells dependent on metabolic homeostasis provided by autophagy. Specifically, the continuous mtDNA synthesis by POLG in starving wild-type cells was inhibited by nucleotide insufficiency and elevated mitochondria-derived reactive oxygen species in the presence of autophagy dysfunction. Moreover, after prolonged starvation, 3'-5' exonuclease-dependent mtDNA degradation by POLG adjusted the initially increasing mtDNA copy number in wild-type cells, but caused quantitative mtDNA instability and irreversible respiratory dysfunction in autophagy-deficient cells as a result of nucleotide limitations. In summary, our study reveals that mitochondria rely on the homeostatic functions of autophagy to balance synthetic and degradative modes of POLG, which control copy number dynamics and stability of the mitochondrial genome.

Figure 1.

Autophagy sustains mtDNA synthesis and stability during starvation. (A and B) mtDNA synthesis depends on autophagy during starvation. WT and Δatg7 cells were grown to log-phase (0 d) or shifted to starvation medium, and DNA synthesis was assessed using EdU staining. (A) Single section images after visualization of EdU incorporation in WT, WT rho⁰, Δatg7, and Δatg7 rho⁰ cells during log-phase (0 d) or starvation (1 d). (B) Quantification of nDNA and mtDNA synthesis during log-phase (0 d) and starvation (1–3 d) in WT and Δatg7 cells. Data are means ± SD (n ≥ 3; 150 cells). (C–E) Defective autophagy causes mtDNA depletion during starvation. (C) WT and Δatg7 cells were grown to log-phase and shifted to starvation medium. mtDNA foci were visualized by DAPI staining and in vivo fluorescence imaging at indicated time points. Single section images are shown. (D and E) Quantification of cells with mtDNA foci or the number of mtDNA foci in foci-positive cells. Data are means ± SD (n = 3; ≥75 cells). (F) mtDNA copy number dynamics in dependence of autophagy during starvation. Quantitative PCR was performed on isolated DNA from WT and Δatg7 cells at indicated time points after starvation. Data are normalized to mtDNA copy number of WT at 0 d set as 1. Data are means ± SD (n = 6). (G) Respiratory deficiency upon regrowth after starvation of WT and Δatg7 cells at indicated time points. Data are means ± SD (n = 3). Dashed lines indicate cell boundaries. Bars, 2 µm. t tests: *, P < 0.05; ***, P < 0.001. Rel., relative.

Figure 2.

Nucleotide availability limits mtDNA stability in autophagy-deficient cells during starvation. (A and B) mtDNA synthesis in dependence of nucleotide levels. WT, Δsml1, Δatg7, and Δsml1Δatg7 cells or WT and Δatg7 cells supplemented with nucleobases were assessed after 1 d of starvation as described in Fig. 1 (A and B). Data are means ± SD (n ≥ 3; 150 cells). (C–E) Increased nucleotide levels stabilize mtDNA in the absence of autophagy. WT, Δsml1, Δatg7, and Δsml1Δatg7 cells or WT and Δatg7 cells supplemented with nucleobases were analyzed by DAPI staining and fluorescence imaging as described in Fig. 1 (C–E). Data are means ± SD (n = 3; ≥75 cells). (F) Increased nucleotide levels stabilize mtDNA in autophagy-deficient cells during starvation. Cells were treated as described in C and analyzed by quantitative PCR as described in Fig. 1 F. Data are means ± SD (n ≥ 3). (G) Increased nucleotide levels do not sustain functional integrity of mtDNA in autophagy mutants. Cells treated as described in C were analyzed as described in Fig. 1 G. Data are means ± SD (n = 3). Dashed lines indicate cell boundaries. Bars, 2 µm. t tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Rel., relative.

Figure 3.

Elevated ROS level and nucleotide insufficiency inhibit mtDNA synthesis and copy number maintenance in autophagy-deficient cells during starvation. (A) Defective autophagy causes complex III–dependent elevated ROS production during starvation. WT, Δcbs1, Δatg7, and Δcbs1Δatg7 cells were grown in log-phase (0 d) or shifted to starvation media ± nucleobases and analyzed for ROS levels using DHE staining and flow cytometry at indicated time points. Data are means ± SD (n = 3). (B and C) mtDNA synthesis in autophagy-deficient cells requires ROS reduction and increased nucleotides. WT, Δcbs1, Δatg7, and Δcbs1Δatg7 cells were treated as described in A and assessed after 1 d of starvation as described in Fig. 1 A. Data are means ± SD (n ≥ 3; 150 cells). (D–F) mtDNA maintenance in autophagy-deficient cells in dependence of ROS production and nucleotide supplementation. WT, Δcbs1, Δatg7, and Δcbs1Δatg7 cells were treated as described in A and analyzed at indicated time points as described in Fig. 1 (C–E). (E and F) Quantification of cells showing mtDNA foci or their number of mtDNA foci. Data are means ± SD (n = 3; ≥75 cells). (G) ROS reduction and nucleotide supplementation synergistically stabilize mtDNA copy number in autophagy-deficient cells. WT, Δcbs1, Δatg7, and Δcbs1Δatg7 cells were treated as described in D and analyzed as described in Fig. 1 F. Data are means ± SD (n = 6). Dashed lines indicate cell boundaries. Bars, 2 µm. t tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Rel., relative.

Figure 4.

Exonuclease activity of POLG regulates mtDNA copy number in WT and is required for mtDNA degradation in autophagy mutants during starvation. (A and B) mtDNA synthesis is unaffected by exonuclease-activity of POLG. POLG, Δatg7 POLG, polg^exo−, and Δatg7 polg^exo− cells were treated as described in Fig. 1 A. Data are means ± SD (n ≥ 3; 150 cells). (C–E) Exonuclease-deficiency of POLG rescues mtDNA maintenance in autophagy-deficient cells during starvation. POLG, Δatg7 POLG, polg^exo−, and Δatg7 polg^exo− cells were grown and analyzed as described in Fig. 1 (C–E). Data are means ± SD (n = 3; ≥75 cells). (F) Exonuclease deficiency of POLG prevents decrease in mtDNA copy number during starvation. POLG, Δatg7 POLG, polg^exo−, and Δatg7 polg^exo− cells were analyzed as described in Fig. 1 F. Data are means ± SD (n = 3). Dashed lines indicate cell boundaries. Bars, 2 µm. t tests: *, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant. Rel., relative.

Figure 5.

Model for mtDNA copy number regulation by POLG in dependence of autophagy. POLG regulates mtDNA copy number by balanced DNA synthesis and degradation. During starvation, autophagy provides metabolites to fuel nucleotide pools (dNTPs) and prevent elevated ROS production in mitochondria, which is required for sustained mtDNA synthesis and suppressed mtDNA degradation by POLG.