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Updated: Jul 18, 2025
Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
Published on: August 21, 2021
Maintenance of Flap Endonucleases for Long-Patch Base Excision DNA Repair in Mouse Muscle and Neuronal Cells
Rachel A Caston1, Paola Fortini2, Kevin Chen1
1Department of Pharmacological Sciences, Renaissance School of Medicine, Stony Brook University, Stony Brook, NY 11794, USA.
Abstract:
After cellular differentiation, nuclear DNA is no longer replicated, and many of the associated proteins are downregulated accordingly. These include the structure-specific endonucleases Fen1 and DNA2, which are implicated in repairing mitochondrial DNA (mtDNA). Two more such endonucleases, named MGME1 and ExoG, have been discovered in mitochondria. This category of nuclease is required for so-called "long-patch" (multinucleotide) base excision DNA repair (BER), which is necessary to process certain oxidative lesions, prompting the question of how differentiation affects the availability and use of these enzymes in mitochondria. In this study, we demonstrate that Fen1 and DNA2 are indeed strongly downregulated after differentiation of neuronal precursors (Cath.a-differentiated cells) or mouse myotubes, while the expression levels of MGME1 and ExoG showed minimal changes. The total flap excision activity in mitochondrial extracts of these cells was moderately decreased upon differentiation, with MGME1 as the predominant flap endonuclease and ExoG playing a lesser role. Unexpectedly, both differentiated cell types appeared to accumulate less oxidative or alkylation damage in mtDNA than did their proliferating progenitors. Finally, the overall rate of mtDNA repair was not significantly different between proliferating and differentiated cells. Taken together, these results indicate that neuronal cells maintain mtDNA repair upon differentiation, evidently relying on mitochondria-specific enzymes for long-patch BER.
Insights
Cellular differentiation downregulates some DNA repair enzymes but not others in mitochondria. Neuronal cells maintain mitochondrial DNA repair, using specific enzymes for base excision repair.
Area of Science:
- Mitochondrial biology
- DNA repair mechanisms
- Cellular differentiation
Background:
- Nuclear DNA replication ceases after differentiation, leading to protein downregulation.
- Structure-specific endonucleases like Fen1 and DNA2 are involved in mitochondrial DNA (mtDNA) repair.
- Mitochondria possess unique endonucleases, MGME1 and ExoG, crucial for long-patch base excision repair (BER).
Purpose of the Study:
- To investigate the impact of cellular differentiation on mitochondrial DNA repair enzymes.
- To determine how differentiation affects the activity and expression of specific endonucleases in mitochondria.
- To understand the maintenance of mtDNA integrity during neuronal and muscle cell differentiation.
Main Methods:
- Comparing expression levels of Fen1, DNA2, MGME1, and ExoG in proliferating versus differentiated neuronal and muscle cells.
- Assessing total flap excision activity in mitochondrial extracts from these cells.
- Quantifying oxidative and alkylation damage in mtDNA.
- Measuring the overall rate of mtDNA repair.
Main Results:
- Fen1 and DNA2 were significantly downregulated post-differentiation, while MGME1 and ExoG showed minimal changes.
- Total mitochondrial flap excision activity decreased moderately, with MGME1 being the primary enzyme.
- Differentiated cells accumulated less oxidative/alkylation damage in mtDNA compared to proliferating cells.
- The overall rate of mtDNA repair remained consistent between proliferating and differentiated cells.
Conclusions:
- Neuronal and muscle cells maintain mtDNA repair capacity after differentiation.
- Mitochondria-specific enzymes, particularly MGME1, are key for long-patch BER in differentiated cells.
- Despite changes in specific enzyme levels, overall mtDNA repair efficiency is preserved.
