Dataset Information

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

ABSTRACT: While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.

SUBMITTER: Chesner LN

PROVIDER: S-EPMC7855827 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

Chesner Lisa N LN Essawy Maram M Warner Cecilia C Campbell Colin C

DNA repair 20201125

While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasm ...[more]

PMID: 33316746

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Project description:Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Pol?) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Pol?-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Pol?-DPC accumulation and trapped ubiquitin in the DPC, with Pol? accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase ? (Pol?), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Pol?, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Pol?: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H2O2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H2O2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Pol?, and OGG1 proteins. As seen for Pol?, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Pol?-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells.

Dataset Information

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

Publications

DNA-protein crosslinks are repaired via homologous recombination in mammalian mitochondria.

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