DNA recombination in plant mitochondria: Identification and functional analysis of a mitochondrial targeted homologue of a bacterial RecA protein in Arabidopsis thaliana

The homologous recombination (HR) mechanism has an important role in generating genetic variability among living organisms by constant rearrangement and exchange of DNA within and between genomes. Functional homologues of RecA, a key enzyme of HR, have been identified and characterized in all eubacteria and almost all eukaryota, including higher plants. Very little is known about HR in other genetic systems like the chloroplast or mitochondria, in contrast to the vast amount of knowledge accumulated on nuclear HR. While HR in yeast mitochondria has been clearly demonstrated, bacteria-like recombination activity has recently been shown in plant chloroplasts and human mitochondria. The common ancestral links of chloroplasts and mitochondria to primitive bacteria suggest the possibility of a bacteria-like HR mechanism in plant mitochondria.; In Glycine max (soy bean), we have identified complex recombination intermediates and single-stranded DNA ends in mitochondrial DNA (mtDNA) samples analyzed by 2-dimensional gel electrophoresis and electron microscopy. We have also identified strand transfer activity in protein fractions from mitochondrial extracts. The strand exchange activity was found to be dependent on ATP hydrolysis, suggesting the involvement of a RecA-like protein in catalyzing this reaction.; We have identified in Arabidopsis thaliana, a 43 kDa nuclear encoded mitochondrial protein that is immunologically related to chloroplast RecA and shares high homology with RecA proteins from the members of the primitive alpha-proteobacteria subdivision and eukaryotic homologues of RecA. In vitro targeting experiments showed that the presequence from the mt-recA localizes green fluorescent protein (GFP) marker specifically into isolated mitochondria when the presequence was cloned 5 ' to the GFP coding sequence to generate a N-terminal transit peptide. The mitochondrial recA (mt-recA) gene was overexpressed in E. coli and the purified recombinant protein was used to show strand exchange of homologous DNA substrates in vitro. When an expression vector carrying the mt-recA gene was transformed into an E. coli host carrying a recA deletion, the mt-recA was able to partially complement the deleterious effects of DNA damaging agents. These results provide the first biochemical evidence on proteins involved in mitochondrial DNA recombination in plants. This information could be useful for development of mitochondrial transformation technology and engineering crops with better genotypes and phenotypes.