Mechanisms of mitochondrial DNA deletion formation - The fate of linear mtDNA
Molecular Biology and Physiology of Neurons and Glial Cells
Final Report Abstract
Maintenance of the circular mitochondrial genome is of key importance for sufficient energy supply of the cell provided by mitochondria. Replication errors or external damage can cause strand breaks, thus, eventually generate linear species of the mitochondrial DNA (mtDNA). Such strand breaks might be repaired by enzymatic pathways that are similar to those acting in the nucleus. Since mtDNA is present in multiple copies in most cells, an alternative option is to eliminate damaged mtDNA molecules and replace them by replication of intact mtDNA. Some potential players in the repair of mtDNA has previously been proposed, but nothing was known about the molecular machinery underlying mtDNA degradation. Our initial observation that patients’ cells deficient for the mitochondrial MGME1 exonuclease accumulate linear mtDNA species prompted us to investigate the role of this and other candidate enzymes in mtDNA degradation. We generated knockout and mutated HEK293 cell lines using the CRISPR–Cas9 technology and exposed them to different conditions of mtDNA damage. (a) In order to investigate the mtDNA degradation machinery, we used cells in which mitochondrial-targeted restriction endonucleases introduced uniform double-strand breaks in a doxycycline-inducible manner. (b) We treated cells with hydrogen peroxide to mimic acute oxidative damage occurring within the body. (c) In order to reduce the oxidative stress experienced by cells cultured under regular conditions, we also grew cells in low oxygen tension atmosphere. Combining Southern blotting, single-molecule PCR, and ultra-deep next-generation sequencing of isolated mtDNA, we were able to identify (1) three major constituents of the mtDNA degradation machinery: the MGME1 5’-to-3’ exonuclease, the 3’-to-5’ exonuclease activity of the replicative DNA polymerase POLG, and the TWNK helicase. Importantly, these enzymes are also key components of the mtDNA replication complex. (2) We observed that ongoing mtDNA degradation results in ends that are proximal to GC-stretches suggesting that the degradation process preferentially pauses at such sites. (3) Acute oxidative damage produces abundant double-strand breaks and the subsequent processing of linear mtDNA in wild-type cells results in ends that resemble degradation intermediates. Intact supercoiled circular mtDNA is recovered within 4–6 hours in wild-type cells, but not in MGME1 and LIG3 knockout cells or in cells carrying exonuclease deficient POLG. (4) Low-oxygen environment leads to the downregulation of mtDNA copy numbers, which is at least partially regulated by the HIF1A transcription factor. Expression of MGME1 is upregulated during this process and MGME1- deficient cells show a weakened hypoxia-induced depletion, which suggest that MGME1 also plays a role in mtDNA copy number regulation. Our results shed light to mtDNA degradation as a new aspect of mtDNA quality control. Methods established in the frameworks of this project and the created cellular models will be valuable tools for further investigation of the interplay between repair and degradation of the mitochondrial genome. This will help us to better understand disease mechanisms related to mtDNA maintenance as well as to promote the development of raising gene therapy approaches based on targeted cleavage and subsequent elimination of mutated mtDNA.
Publications
- (2018) Is There Still Any Role for Oxidative Stress in Mitochondrial DNA-Dependent Aging? Genes (Basel) 9:175
Zsurka G, Peeva V, Kotlyar A, Kunz WS
(See online at https://doi.org/10.3390/genes9040175) - (2018) Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun 9:1727
Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS
(See online at https://doi.org/10.1038/s41467-018-04131-w) - (2019) Distinct segregation of the pathogenic m.5667G>A mitochondrial tRNA-Asn mutation in extraocular and skeletal muscle in chronic progressive external ophthalmoplegia. Neuromuscul Disord 29:358–367
Schlapakow E, Peeva V, Zsurka G, Jeub M, Wabbels B, Kornblum C, Kunz WS
(See online at https://doi.org/10.1016/j.nmd.2019.02.009) - (2019) Replication fork rescue in mammalian mitochondria. Sci Rep 9:8785
Torregrosa-Muñumer R, Hangas A, Goffart S, Blei D, Zsurka G, Griffith J, Kunz WS, Pohjoismäki JLO
(See online at https://doi.org/10.1038/s41598-019-45244-6) - (2021) Molecular and functional effects of loss of cytochrome c oxidase subunit 8A. Biochemistry (Mosc) 86:33–43
Rotko D, Kudin AP, Zsurka G, Kulawiak B, Szewczyk A, Kunz WS
(See online at https://doi.org/10.1134/s0006297921010041)
