Expression of E. coli RecA targeted to mitochondria of human cells.
Mitochondrial DNA integrity is ensured by several nuclear-encoded proteins in vertebrates, and a number of mtDNA alterations in human diseases, including deletions and duplications, have been suspected to result from errors in the mitochondrial recombination pathway. However, the presence of the latter system is still a matter of controversy as RecA proteins display various functions in vitro. In Escherichia coli, RecA plays a central role in homologous recombination by pairing and transferring a single strand to a homologous duplex DNA. To address indirectly the issue of a mitochondrial recombination pathway in vivo, we have constructed a chimeric gene containing an N terminal mitochondrial targeting sequence and the E. coli RecA gene. Cells were transfected by the recombinant plasmid, then tested for their mtDNA repair upon bleomycin treatment. We found an increased repair rate of the mitochondrial DNA in cells expressing RecA as compared to control cells. These results indicate that the transfected cells display an improved mtDNA repair replication pathway due to the exogenous RecA, likely in synergy with an endogenous rate-limiting mitochondrial recombination pathway.
Paul
R
R
EMI 99.29 INSERM, Génétique Mitochondriale, Université Victor Segalen Bordeaux 2, 146 rue Léo Saignat, 33076 Cedex, Bordeaux, France.
Dalibart
R
R
Lemoine
S
S
Lestienne
P
P
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001761:Bleomycin / Q000494:pharmacology; D015139:Blotting, Southern; D015153:Blotting, Western; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D004272:DNA, Mitochondrial / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D011938:Rec A Recombinases / Q000096:biosynthesis*; D011995:Recombination, Genetic; D014158:Transcription, Genetic; D014162:Transfection
D000900:Anti-Bacterial Agents; D004272:DNA, Mitochondrial; D001761:Bleomycin; D011938:Rec A Recombinases