INHIBITION OF CHLOROPLAST DNA RECOMBINATION AND REPAIR BY DOMINANT-NEGATIVE MUTANTS OF ESCHERICHIA-COLI RECA

The occurrence of homologous DNA recombination in chloroplasts is well documented, but little is known about the molecular mechanisms involv ed or their biological significance. The endosymbiotic origin of plast ids and the recent finding of an Arabidopsis nuclear gene, encoding a chloroplast-localized protein homologous to Escherichia coli RecA, sug gest that the plastid recombination system is related to its eubacteri al counterpart. Therefore, we examined whether dominant negative mutan ts of the E. coli RecA protein can interfere with the activity of thei r putative homolog in the chloroplast of the unicellular green alga Ch lamydomonas reinhardtii. Transformants expressing these mutant RecA pr oteins showed reduced survival rates when exposed to DNA-damaging agen ts, deficient repair of chloroplast DNA, and diminished plastid DNA re combination. These results strongly support the existence of a RecA-me diated recombination system in chloroplasts. We also found that the wi ld-type E. coli RecA protein enhances the frequency of plastid DNA rec ombination over 15-fold, although it has no effect on DNA repair or ce ll survival. Thus, chloroplast DNA recombination appears to be limited by the availability of enzymes involved in strand exchange rather tha n by the level of initiating DNA substrates. Our observations suggest that a primary biological role of the recombination system in plastids is in the repair of their DNA, most likely needed to cope with damage due to photooxidation and other environmental stresses. This hypothes is could explain the evolutionary conservation of DNA recombination in chloroplasts despite the predominantly uniparental inheritance of the ir genomes.