There are many checkpoints that inhibit cell cycle progression in response to defects in inheritance of the nucleus including checkpoints that monitor nuclear DNA duplication, quality control and segregation. We identified a checkpoint that responds to defects in organelle inheritance, and more specifically to loss of mitochondrial DNA (mtDNA). The checkpoint for damage to nuclear DNA does not respond to loss of specific genes encoded by nuclear DNA. Rather, it responds to broader issues, including double strand DNA breaks or stalled replication forks. We find that the mtDNA inheritance checkpoint also does not respond to loss of genes encoded by mtDNA. Rather, it responds to loss of DNA in the organelle. All well-characterized checkpoints have 1) sensors that detect defects in critical cell cycle events, 2) signal transduction machinery to arrest the cell cycle and promote repair, and 3) mechanisms to inactivate the checkpoint. We obtained evidence for a role of Mip1p, mtDNA polymerase ?, and contact sites between mitochondrial outer and inner membranes in sensing loss of mtDNA. We also find that Rad53p, the yeast homologue of the tumor suppressor and the DNA damage checkpoint kinase Chk2, regulates G1 to S progression in response to loss of mtDNA. Our studies reveal the existence of an organelle inheritance checkpoint and raise the possibility that there are checkpoints for the inheritance of other organelles. Since mutation of Chk2 and its upstream (ATM) and downstream (p53) regulators also results in changes in mtDNA content and cell cycle delays in mammalian and human cells, this checkpoint likely exists in other eukaryotes. We will study how information regarding mtDNA content is transmitted from the mitochondrial inner membrane (where mtDNA and Mip1p reside) across contact sites to the cytosol and ultimately to the nucleus (where Rad53p resides). Here, we will focus on the replisome, a protein complex that contains Mip1p, spans mitochondrial contact sites and binds directly to mtDNA, and on pathways to transmit signals from the replisome to Rad53p. Since DNA replication is template-dependent, cells that have lost mtDNA cannot replace it. Therefore, they must adapt to the checkpoint and resume cell cycling to survive. We will study the role of largely uncharacterized genes that we have implicated checkpoint adaptation and regulation of G1 to S progression. The proposed studies will be the first to determine the mechanism underlying the mtDNA inheritance checkpoint and adaptation to that checkpoint. They will also extend our understanding of proteins that interact with mtDNA, particularly at mitochondrial contact sites;mechanisms for regulation of G1 to S progression;and signal transduction in the DNA damage checkpoint. Finally, since the DNA damage checkpoint is the target for mutation in cancer, and mtDNA polymerase ? is the target for mutation in >125 diseases, the studies will extend our understanding of cancer biology and diseases associated with defects in mitochondrial function.
Checkpoints are pathways that block cell replication when critical events that occur during cell division are compromised. They also activate pathways to repair the damage and to trigger cell death when repair is not possible. We identified a new checkpoint that responds to loss of DNA within mitochondria, organelles within cells that are responsible for energy production. Proteins implicated in this process include targets for mutation in cancer and in diseases that affect mitochondrial function. We will study the mechanism of action of these and other proteins in the mitochondrial DNA inheritance checkpoint.
|Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R et al. (2016) Imaging of the Actin Cytoskeleton and Mitochondria in Fixed Budding Yeast Cells. Methods Mol Biol 1365:63-81|
|Higuchi-Sanabria, Ryo; Garcia, Enrique J; Tomoiaga, Delia et al. (2016) Characterization of Fluorescent Proteins for Three- and Four-Color Live-Cell Imaging in S. cerevisiae. PLoS One 11:e0146120|
|Higuchi-Sanabria, Ryo; Charalel, Joseph K; Viana, Matheus P et al. (2016) Mitochondrial anchorage and fusion contribute to mitochondrial inheritance and quality control in the budding yeast Saccharomyces cerevisiae. Mol Biol Cell 27:776-87|
|Higuchi-Sanabria, Ryo; Swayne, Theresa C; Boldogh, Istvan R et al. (2016) Live-Cell Imaging of Mitochondria and the Actin Cytoskeleton in Budding Yeast. Methods Mol Biol 1365:25-62|
|Pernice, Wolfgang M; Vevea, Jason D; Pon, Liza A (2016) A role for Mfb1p in region-specific anchorage of high-functioning mitochondria and lifespan in Saccharomyces cerevisiae. Nat Commun 7:10595|
|Vevea, Jason D; Garcia, Enrique J; Chan, Robin B et al. (2015) Role for Lipid Droplet Biogenesis and Microlipophagy in Adaptation to Lipid Imbalance in Yeast. Dev Cell 35:584-99|
|Higuchi-Sanabria, Ryo; Pernice, Wolfgang M A; Vevea, Jason D et al. (2014) Role of asymmetric cell division in lifespan control in Saccharomyces cerevisiae. FEMS Yeast Res 14:1133-46|
|Vevea, Jason D; Swayne, Theresa C; Boldogh, Istvan R et al. (2014) Inheritance of the fittest mitochondria in yeast. Trends Cell Biol 24:53-60|
|Wolken, Dana M Alessi; McInnes, Joseph; Pon, Liza A (2014) Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast. Mol Biol Cell 25:753-62|
|Pon, Liza A (2013) Mitochondrial fission: rings around the organelle. Curr Biol 23:R279-81|
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