Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress.
Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (>48h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity.
Dept. 75, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, Czech Republic. Electronic address: lukas.alan@fgu.cas.cz.
Lukáš
L
Alán
Dept. 75, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, Czech Republic.
Tomáš
T
Špaček
Dept. 75, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, Czech Republic.
David
D
Pajuelo Reguera
Dept. 75, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, Czech Republic.
Martin
M
Jabůrek
Dept. 75, Institute of Physiology, Academy of Sciences of the Czech Republic v.v.i, Czech Republic.
Petr
P
Ježek
D004249:DNA Damage; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004317:Doxorubicin; D034281:Dynamins; D004996:Ethidium; D020558:GTP Phosphohydrolases / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; C502070:MAP1LC3B protein, human; D008869:Microtubule-Associated Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; C504091:NABP2 protein, human; C499631:TFAM protein, human; C438387:TIMM23 protein, human; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D004317:Doxorubicin; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; D034281:Dynamins; D004996:Ethidium
Doxorubicin; Ethidium Bromide; Mitochondrial DNA stress; Mitochondrial transcription factor A; Nucleoid clusters