Selective mitochondrial DNA degradation following double-strand breaks.

Mitochondrial DNA (mtDNA) can undergo double-strand breaks (DSBs), caused by defective replication, or by various endogenous or exogenous sources, such as reactive oxygen species, chemotherapeutic agents or ionizing radiations. MtDNA encodes for proteins involved in ATP production, and maintenance of genome integrity following DSBs is thus of crucial importance. However, the mechanisms involved in mtDNA maintenance after DSBs remain unknown. In this study, we investigated the consequences of the production of mtDNA DSBs using a human inducible cell system expressing the restriction enzyme PstI targeted to mitochondria. Using this system, we could not find any support for DSB repair of mtDNA. Instead we observed a loss of the damaged mtDNA molecules and a severe decrease in mtDNA content. We demonstrate that none of the known mitochondrial nucleases are involved in the mtDNA degradation and that the DNA loss is not due to autophagy, mitophagy or apoptosis. Our study suggests that a still uncharacterized pathway for the targeted degradation of damaged mtDNA in a mitophagy/autophagy-independent manner is present in mitochondria, and might provide the main mechanism used by the cells to deal with DSBs.

DNA maintenance following bleomycin-induced strand breaks does not require poly(ADP-ribosyl)ation activation in Drosophila S2 cells.

BACKGROUND: Poly-ADP ribosylation (PARylation) is a post translational modification, catalyzed by Poly(ADP-ribose)polymerase (PARP) family. In Drosophila, PARP-I (human PARP-1 ortholog) is considered to be the only enzymatically active isoform. PARylation is involved in various cellular processes such as DNA repair in case of base excision and strand-breaks. OBSERVATIONS: Strand-breaks (SSB and DSB) are detrimental to cell viability and, in Drosophila, that has a unique PARP family organization, little is known on PARP involvement in the control of strand-breaks repair process. In our study, strands-breaks (SSB and DSB) are chemically induced in S2 Drosophila cells using bleomycin. These breaks are efficiently repaired in S2 cells. During the bleomycin treatment, changes in PARylation levels are only detectable in a few cells, and an increase in PARP-I and PARP-II mRNAs is only observed during the recovery period. These results differ strongly from those obtained with Human cells, where PARylation is strongly activating when DNA breaks are generated. Finally, in PARP knock-down cells, DNA stability is altered but no change in strand-breaks repair can be observed. CONCLUSIONS: PARP responses in DNA strands-breaks context are functional in Drosophila model as demonstrated by PARP-I and PARP-II mRNA increases. However, no modification of the global PARylation profile is observed during strand-breaks generation, only changes at cellular levels are detectable. Taking together, these results demonstrate that PARylation process in Drosophila is tightly regulated in the context of strands-breaks repair and that PARP is essential during the maintenance of DNA integrity but dispensable in the DNA repair process.

A comprehensive approach to determining BER capacities and their change with aging in Drosophila melanogaster mitochondria by oligonucleotide microarray.

DNA repair mechanisms are key components for the maintenance of the essential mitochondrial genome. Among them, base excision repair (BER) processes, dedicated in part to oxidative DNA damage, are individually well known in mitochondria. However, no large view of these systems in differential physiological conditions is available yet. Combining the use of pure mitochondrial fractions and a multiplexed oligonucleotide cleavage assay on a microarray, we demonstrated that a large range of glycosylase activities were present in Drosophila mitochondria. Most of them were quantitatively different from their nuclear counterpart. Moreover, these activities were modified during aging.

Bleomycin-induced double-strand breaks in mitochondrial DNA of Drosophila cells are repaired.

Mitochondrial DNA lesions cause numerous human diseases, and it is therefore important to identify the mechanisms whereby the mitochondrion repairs the damage. We have studied in cultured Drosophila cells the repair of bleomycin-induced double-strand breaks (DSBs) in mitochondrial DNA. Our results show that DSBs are repaired as rapidly and effectively in the mitochondria as in the nucleus. DNA repair is complete within 2h following bleomycin treatment, showing that Drosophila mitochondria have an effective system of DSB repair. The mechanism and mitochondrial proteins involved remain to be identified.

Coordinated decrease of the expression of the mitochondrial and nuclear complex I genes in a mitochondrial mutant of Drosophila.

We have studied a mutant strain of Drosophila in which 80% of the mitochondrial DNA molecules have lost over 30% of their coding region through deletion. This deletion affects genes encoding five subunits of complex I of the respiratory chain (NADH:ubiquinone oxidoreductase). The enzymatic activity of complex I in the mutant strain is half that in the wild strain, but ATP synthesis is unaffected. The drop in enzymatic activity of complex I in the mutant strain is associated with a 50% decrease in the quantity of constitutive proteins of the complex. Moreover, in the mutant strain there is a 50% decrease in the steady-state concentration of the transcripts of the mitochondrial genes affected by the deletion. This decrease is also observed for the transcripts of the nuclear genes coding for the subunits of complex I. These results suggest a coordination of the expression of the mitochondrial and nuclear genes coding for mitochondrial proteins.

The nuclear genome of a Drosophila mutant strain increases the frequency of rearranged mitochondrial DNA molecules.

We studied a mutant strain of Drosophila subobscura, in which 80% of the mitochondrial genomes (mtDNA) have lost over 30% of the coding region. The mutation is stable and is transmitted identically to offspring. The putative role of the mutant nuclear genome in the production of rearranged mtDNA was investigated using reciprocal crosses, to place the mitochondria of the wild strain in a mutant nuclear context. Nested PCR was used to screen for rearrangements in different regions of mtDNA; and rearrangements were detected in some individuals from the F6 generation. The frequency of these deleted mtDNAs then increased progressively in the population; and they were present in nearly all individuals in the F11 generation. They were not transmissible. Direct repeats were present at the deletion boundaries. These mutated genomes disappeared on reversion to a wild-type nuclear genome. Deletions were detected in a very small fraction of the wild population (0.7% of individuals). The mutant nuclear genome therefore does not promote a particular deletion but increases the frequency of different mtDNA rearrangements. The potential involvement of different candidate nuclear genes is discussed.