Abstract
Endonuclease G (EndoG) is a nuclear-encoded endonuclease, mostly localised in mitochondria. In the nucleus EndoG participates in site-specific cleavage during replication stress and genome-wide DNA degradation during apoptosis. However, the impact of EndoG on mitochondrial DNA (mtDNA) metabolism is poorly understood. Here, we investigated whether EndoG is involved in the regulation of mtDNA replication and removal of aberrant copies. We applied the single-cell mitochondrial Transcription and Replication Imaging Protocol (mTRIP) and PCR-based strategies on human cells after knockdown/knockout and re-expression of EndoG. Our analysis revealed that EndoG stimulates both mtDNA replication initiation and mtDNA depletion, the two events being interlinked and dependent on EndoG's nuclease activity. Stimulation of mtDNA replication by EndoG was independent of 7S DNA processing at the replication origin. Importantly, both mtDNA-directed activities of EndoG were promoted by oxidative stress. Inhibition of base excision repair (BER) that repairs oxidative stress-induced DNA damage unveiled a pronounced effect of EndoG on mtDNA removal, reminiscent of recently discovered links between EndoG and BER in the nucleus. Altogether with the downstream effects on mitochondrial transcription, protein expression, redox status and morphology, this study demonstrates that removal of damaged mtDNA by EndoG and compensatory replication play a critical role in mitochondria homeostasis.
Highlights
Mitochondria harbour their own small circular DNA (16569 bp in size in humans), which encodes 13 proteins, essential for the functionality of the electron transport chain (ETC), 22 tRNAs as well as 2 ribosomal RNAs (Figure 1A). mitochondrial DNA (mtDNA) molecules contain a noncoding region (NCR), where the light and heavy strand promoters (LSP and HSP) as well as the origin of heavy strand replication (OH) are located [1, 2]
The principle of mitochondrial Transcription and Replication Imaging Protocol (mTRIP) is a combination www.oncotarget.com of RNA and DNA fluorescence in situ hybridization (FISH), relying on the following probe design: the mREP probe is localised in the upstream neighbourhood of the main replication origin OH and recognises non-transcribed DNA that becomes accessible during initiation of mtDNA replication; this signal includes the production of extended mtDNA replication as well as abortive 7S structures
The mTRANS probe is a mix of three probes indicating mitochondrial transcription by the labelling of processed mitochondrial RNAs and unprocessed immature mitochondrial RNAs encoded at different positions within the mitochondrial genome
Summary
Mitochondria harbour their own small circular DNA (16569 bp in size in humans), which encodes 13 proteins, essential for the functionality of the electron transport chain (ETC), 22 tRNAs as well as 2 ribosomal RNAs (Figure 1A). mtDNA molecules contain a noncoding region (NCR), where the light and heavy strand promoters (LSP and HSP) as well as the origin of heavy strand replication (OH) are located [1, 2]. To date two prevalent models describe replication in mammalian mitochondria: First, the strand-displacement replication model, which was suggested in the 1970s and postulates that heavy-strand synthesis starts within the displacementloop (D-loop) near or at OH and proceeds unidirectionally until after two thirds the light-strand DNA synthesis is initiated at OL (origin of light strand replication) on the other strand [3] According to this model mtDNA www.oncotarget.com replication results in single-stranded intermediates, which are coated and stabilised by the mitochondrial singlestranded DNA binding protein (mtSSB). The RITOLS (Ribonucleotide Incorporated ThroughOut the Lagging Strand) model proceeds via such heavystrand intermediates, the light-strand becomes temporarily hybridised to RNA, which is subsequently removed prior DNA synthesis [4] According to this last model, -called the bootlace mechanism, this RNA is derived from processed transcripts, threaded onto the displaced light-strand template to aid protecting singlestranded intermediates from damage and nucleolysis [5]. The role of 7S DNA is still unclear [7]
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