Rad53 is essential for a mitochondrial DNA inheritance checkpoint regulating G1 to S progression

Abstract

The Chk2-mediated deoxyribonucleic acid (DNA) damage checkpoint pathway is important for mitochondrial DNA (mtDNA) maintenance. We show in this paper that mtDNA itself affects cell cycle progression. Saccharomyces cerevisiae rho(0) cells, which lack mtDNA, were defective in G1- to S-phase progression. Deletion of subunit Va of cytochrome c oxidase, inhibition of F(1)F(0) adenosine triphosphatase, or replacement of all mtDNA-encoded genes with noncoding DNA did not affect G1- to S-phase progression. Thus, the cell cycle progression defect in rho(0) cells is caused by loss of DNA within mitochondria and not loss of respiratory activity or mtDNA-encoded genes. Rad53p, the yeast Chk2 homologue, was required for inhibition of G1- to S-phase progression in rho(0) cells. Pif1p, a DNA helicase and Rad53p target, underwent Rad53p-dependent phosphorylation in rho(0) cells. Thus, loss of mtDNA activated an established checkpoint kinase that inhibited G1- to S-phase progression. These findings support the existence of a Rad53p-regulated checkpoint that regulates G1- to S-phase progression in response to loss of mtDNA.

Figure 1.

Cell cycle defects are caused by loss of DNA in mitochondria. Wild-type (BY4741) cells containing mtDNA (rho⁺) or cells lacking mtDNA (rho⁰, mgm101Δ cells) were synchronized and propagated at 30°C. At the times shown, cells were fixed and stained with propidium iodide, and their DNA content was assessed by flow cytometry. (A) Images of DAPI-stained cells are 2D projections of deconvolved 3D volumes of rho⁺, rho⁰, and mgm101Δ rho⁰ cells. Cell outlines are shown in white. n, nuclear DNA; m, mtDNA. Bar, 1 µm. (B) Rho⁺ exhibit normal cell cycle progression. Cells lacking mtDNA either by EtBr treatment (rho⁰) or by deletion of a gene required for mtDNA maintenance (mgm101Δ rho⁰) fail to progress normally and show a G1 arrest. (C) Quantitation of progression through G1 phase of rho⁺, rho⁰, and mgm101Δ rho⁰ cells was assessed as the fold change in the fraction of cells in G1 phase at the time specified, relative to the fraction of cells that were in G1 at the time of release from pheromone-induced G1 arrest (cells in G1 at t_x/cells in G1 at t₀). The data shown are pooled from multiple experiments (wild type, n = 5; rho⁰, n = 3; mgm101Δ rho⁰, n = 3).

Figure 2.

Inhibition of respiration or of ATP production does not cause the defect in passage through G1. Cell cycle progression was assessed in cells treated with the F₁F₀ ATPase inhibitor oligomycin and in cells bearing a deletion in a nuclear-encoded mitochondrial respiratory chain component (COX5A). (A) Effect of oligomycin on yeast cell growth. Single colonies were incubated in liquid media with or without 1 µg/ml oligomycin and aerated at 30°C. Cell growth was assessed by measuring the OD₆₀₀ of the culture. Oligomycin-dependent inhibition of yeast cell growth on a nonfermentable carbon source was observed on solid media in three independent experiments. Quantitation of cell density in liquid cultures in the presence and absence of oligomycin was performed once. (B) Cell cycle progression of cells in the absence or presence of oligomycin was performed as for Fig. 1. (C) Quantitation of progression from G1 to S in oligomycin-treated and control cells was performed as for Fig. 1. The data shown are pooled from three experiments. (D) Imaging of DAPI-stained rho⁺, cox5aΔ rho⁺, and cox5aΔ rho⁰ cells was performed as for Fig. 1. Cell outlines are shown in white. n, nuclear DNA; m, mtDNA. Bar, 1 µm. (E) Cell cycle progressions of rho⁺, cox5aΔ rho⁺, and cox5aΔ rho⁰ cells were determined as for Fig. 1. (F) Quantitation of progression from G1 to S in rho⁺, cox5aΔ rho⁺, and cox5aΔ rho⁰ cells was performed as for Fig. 1. The data shown are pooled from multiple experiments (wild type [WT], n = 8; cox5aΔ rho⁺, n = 3; cox5aΔ rho⁰, n = 4).

Figure 3.

Cell cycle defects are caused by loss of DNA in mitochondria. (A) Imaging of DAPI-stained rho⁺, rho⁰, and N24 rho⁻ cells was performed as for Fig. 1. All cells used were in the D273-10B genetic background. Cell outlines are shown in white. n, nuclear DNA; m, mtDNA. Bar, 1 µm. (B) Cell cycle progression of rho⁺, rho⁰, or N24 rho⁻ cells was performed as for Fig. 1. (C) Quantitation of cell cycle progression of rho⁺, rho⁰, and N24 rho⁻ cells was performed as for Fig. 1 C. The data shown are pooled from three experiments.

Figure 4.

Rad53 is required for regulation of cell cycle progression in response to loss of mtDNA. (A) 2D projections of 3D volumes of DAPI-stained sml1Δ rho⁺ and sml1Δ rho⁰ cells. (B) Cell cycle progression of sml1Δ rho⁺ and sml1Δ rho⁰ cells was assessed as for Fig. 1. (C) Quantitation of cell cycle progression of sml1Δ rho⁺ and sml1Δ rho⁰ cells was performed as for Fig. 1. The data shown are pooled from three experiments. (D) 2D projections of a 3D volume of DAPI-stained rad53Δ sml1Δ rho⁺ and rad53Δ sml1Δ rho⁰ cells. (E) Cell cycle progression of rad53Δ sml1Δ rho⁺ and rad53Δ sml1Δ rho⁰ cells was assessed as for Fig. 1. (F) Quantitation of cell cycle progression of rad53Δ sml1Δ rho⁺ and rad53Δ sml1Δ rho⁰ cells was performed as for Fig. 1. All cells used are from the W303 genetic background. The data shown are pooled from three experiments. Cell outlines are shown in white. n, nucleus; m, mtDNA; WT, wild type. Bars, 1 µm.

Figure 5.

Pif1p undergoes Rad53-dependent phosphorylation in response to loss of mtDNA. (A) Western blot showing myc-tagged Pif1p in rho⁺, rho⁰, and HU-treated rho⁺ cells incubated in the presence (+) or absence (−) of 400 U/ml calf intestinal phosphatase (CIP) for 2 h at 37°C. Hexokinase (HXK) was used as a loading control. (B) Western blots showing myc-tagged Pif1p in rho⁰ cells and in rho⁰ cells bearing deletions in RAD53 and SML1. Asterisks mark phosphorylated Pif1p, which is present in rho⁰ cells and HU-treated rho⁺ cells but not in rad53Δ sml1Δ rho⁰ cells.