Abstract

Mitochondrial protein extracts from normal and immortalized mammalian somatic cells catalyze homologous recombination of plasmid DNA substrates. Mitochondrial homologous recombination activity required exogenous adenosine triphosphate, although substantial activity remained when non-hydrolyzable analogs were used instead. There was no requirement for added nucleoside triphosphates, and the reaction was not inhibited by dideoxyadenosine triphosphate or aphidicolin. The majority of recombinant plasmid molecules result from a conservative process, indicating that nuclease-mediated strand-annealing is not responsible for the mitochondrial homologous recombination activity. Affinity-purified anti-recA antibodies inhibited the reaction, suggesting that activity is dependent on a mammalian mitochondrial homolog of the bacterial strand-transferase protein. The presence of homologous recombination activity within mammalian mitochondrial extracts suggests that this process is involved in mitochondrial DNA repair.

Highlights

  • Mutations in mitochondrial DNA are important for several additional reasons as well

  • When homologous DNA recombination (HR) activity was examined in these extracts, we found that 3-hydroxybutyrate enhanced the specific HR activity in these extracts by 3.3-fold when compared to mitochondrial extracts prepared from cells not supplemented with 3-hydroxybutyrate (Fig. 4)

  • Previous results have shown that mammalian mitochondria can repair cisplatin inter-strand DNA cross-links [24]

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Summary

Mammalian Mitochondria Possess Homologous DNA Recombination Activity*

Similar studies have shown that mitochondria can repair DNA inter-strand cross-links caused by cisplatin [24], as well as DNA damage caused by bleomycin [25], and the reactive oxygen species-generating compound alloxan [26] These results suggest that while mammalian mitochondria lack nucleotide excision repair activity, they are likely to possess a number of distinct DNA repair pathways. The majority of recombinant clones analyzed are the products of a conservative recombination reaction, strongly suggesting that authentic strand-transferase activity, rather than a nonspecific nuclease-mediated process is responsible for their creation This hypothesis is supported by the observation that pretreatment of extracts with affinity-purified anti-recA antibody reduced homologous recombination activity by approximately 90%. Sess the enzymatic machinery needed to catalyze HR, and suggest that recombinational DNA repair may occur within the mitochondria of mammalian cells

EXPERIMENTAL PROCEDURES
RESULTS
Rat liver mitochondria preparation number
Rat liver
Assay conditions
DISCUSSION
Full Text
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