R-Loop control and mitochondria genome stability requires the 5'-3' exonuclease/flap-endonuclease OEX1

Maintenance of the plant organelle genomes involves factors mostly inherited from their bacterial symbiotic ancestors. In bacteria, a major player in genome maintenance is DNA Polymerase I (Pol I), which provides a 5-3-exonuclease/flap-endonuclease activity required for multiple replication and repair functions. In plant organelles, DNA polymerases POL1A and POL1B are evolutionarily derived from DNA Pol I but lack this domain. In Arabidopsis, OEX1 and OEX2 (Organellar Exonucleases 1 and 2) represent this missing domain and are targeted to mitochondria and chloroplasts, respectively. An oex1 mutant allele shows developmental and fertility defects that correlate with the differential segregation of mitochondrial DNA (mtDNA) subgenomes generated by recombination, suggesting that OEX1 processes replication and recombination intermediates whose accumulation results in genome instability. Alternative splicing generates two OEX1 isoforms that can differentially interact with POL1A and POL1B and variably affect mtDNA repair. Recombinant OEX1 has 5-3-exonuclease and flap endonuclease activities, the latter being a key function in replication and repair. Furthermore, OEX1 has high affinity for RNA:DNA hybrids, rapidly degrading RNA in Okazaki-like structures and R-loops. Consistent with a role in suppressing R-loops, oex1 plants accumulate R-loops in highly transcribed mtDNA regions. Taken together, our results show that OEX1 plays multiple important roles in the processes required to maintain mtDNA stability.