학술논문
The Fate of Oxidative Strand Breaks in Mitochondrial DNA
Document Type
Academic Journal
Author
Source
Antioxidants. May 2023, Vol. 12 Issue 5
Subject
Language
English
ISSN
2076-3921
Abstract
Author(s): Genevieve Trombly [1]; Afaf Milad Said [1]; Alexei P. Kudin [1]; Viktoriya Peeva [1]; Janine Altmüller [2,3]; Kerstin Becker [2,4]; Karl Köhrer [4]; Gábor Zsurka (corresponding author) [1,5,*]; Wolfram [...]
Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H[sub.2]O[sub.2] pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H[sub.2]O[sub.2] pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2–6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H[sub.2]O[sub.2]-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.
Mitochondrial DNA (mtDNA) is particularly vulnerable to somatic mutagenesis. Potential mechanisms include DNA polymerase γ (POLG) errors and the effects of mutagens, such as reactive oxygen species. Here, we studied the effects of transient hydrogen peroxide (H[sub.2]O[sub.2] pulse) on mtDNA integrity in cultured HEK 293 cells, applying Southern blotting, ultra-deep short-read and long-read sequencing. In wild-type cells, 30 min after the H[sub.2]O[sub.2] pulse, linear mtDNA fragments appear, representing double-strand breaks (DSB) with ends characterized by short GC stretches. Intact supercoiled mtDNA species reappear within 2–6 h after treatment and are almost completely recovered after 24 h. BrdU incorporation is lower in H[sub.2]O[sub.2]-treated cells compared to non-treated cells, suggesting that fast recovery is not associated with mtDNA replication, but is driven by rapid repair of single-strand breaks (SSBs) and degradation of DSB-generated linear fragments. Genetic inactivation of mtDNA degradation in exonuclease deficient POLG p.D274A mutant cells results in the persistence of linear mtDNA fragments with no impact on the repair of SSBs. In conclusion, our data highlight the interplay between the rapid processes of SSB repair and DSB degradation and the much slower mtDNA re-synthesis after oxidative damage, which has important implications for mtDNA quality control and the potential generation of somatic mtDNA deletions.