Abstract
Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Mutations in the mitochondrial genome maintenance exonuclease 1 (MGME1) gene were recently reported in mitochondrial disease patients. Here, to study disease pathophysiology, we generated Mgme1 knockout mice and report that homozygous knockouts develop depletion and multiple deletions of mtDNA. The mtDNA replication stalling phenotypes vary dramatically in different tissues of Mgme1 knockout mice. Mice with MGME1 deficiency accumulate a long linear subgenomic mtDNA species, similar to the one found in mtDNA mutator mice, but do not develop progeria. This finding resolves a long-standing debate by showing that point mutations of mtDNA are the main cause of progeria in mtDNA mutator mice. We also propose a role for MGME1 in the regulation of replication and transcription termination at the end of the control region of mtDNA.
Highlights
Replication of mammalian mitochondrial DNA is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function
The vast majority of mutations causing human mitochondrial DNA (mtDNA) instability map to genes encoding proteins involved in mtDNA replication, e.g., the catalytic subunit of mtDNA polymerase (POLGA)[3], the accessory subunit of mtDNA polymerase (POLGB)[4], the replicative helicase (TWNK)[5], DNA replication helicase/nuclease 2 (DNA2)[6], mitochondrial genome maintenance exonuclease 1 (MGME1)[7] and ribonuclease H1 (RNASEH1)[8], or nucleotide pool regulation, e.g., thymidine phosphorylase (TP)[9], mitochondrial thymidine kinase (TK2)[10], deoxyguanosine kinase (DGUOK)[11], ATP-dependent succinate-CoA ligase (SUCLA2)[12], and GTP-dependent succinate-CoA ligase (SUCLG1)[1,13,14]
Reverse transcription (RT)-PCR analyses of the Mgme[1] mRNA confirmed the absence of sequences corresponding to exon 3 (Fig. 1b) and the MGME1 protein was absent on western blots (Fig. 1c) in all investigated tissues of Mgme1−/−mice
Summary
Replication of mammalian mitochondrial DNA (mtDNA) is an essential process that requires high fidelity and control at multiple levels to ensure proper mitochondrial function. Loss of MGME1 expression, either in siRNA treated cells or in patient fibroblasts, leads to an accumulation of 7S DNA7,23, which is the single-stranded DNA species formed by premature replication termination at the end of the control region of mtDNA23, suggesting a role for MGME1 in repressing formation or increasing turnover of these molecules. We have studied the in vivo mtDNA replication phenotypes associated with MGME1 deficiency in various mouse tissues of knockout mice.
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