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Understanding the mechanism behind mtDNA degradation and its significance in maintaining the stability of the mitochondrial genome remains a pivotal and unanswered question in the realm of cell biology. Normally, damaged DNA molecules are only a small fraction of the total mtDNA in a cell, which can be easily removed through the replication process to replace them with intact mtDNA. The concept of a 'disposable genome' holds crucial implications for innovative gene therapy approaches aiming to mitigate mitochondrial DNA diseases. In a previous study, it was reported that the principal protein factors responsible for the rapid degradation of linear mtDNA are also well-known components of the mtDNA replication machinery, including POLG, Twinkle, and MGME1. Mutations in these proteins can lead to mtDNA damage during replication, resulting in a range of disorders such as progressive external ophthalmoplegia (PEO), Perrault syndrome (PS), Pearson marrow-pancreas syndrome (PMPS), and Kearns–Sayre syndrome (KSS).
This study presents a novel method called NISP (Nuclease-Induced Stepwise Photodropping), developed to analyze the degradation of single-stranded DNA by exonucleases and endonucleases. The method allows real-time observation of DNA degradation with high precision and without modifying the enzyme. Using fluorescence-labeled DNA substrates, NISP can measure the activity of enzymes like MGME1 and mung bean nuclease, providing insights into their catalytic behaviors. This research demonstrates how NISP can be applied to study the kinetics of DNA degradation, offering a powerful tool for understanding molecular-level enzyme functions critical in DNA replication, repair, and degradation.
Nucleic Acids Research, gkae822
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