Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomologymediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.DNA repair | evolution | homing endonuclease | I-CreII | plastid DNA C hloroplast DNA (cpDNA) is closely associated with the photosynthetic membranes that harvest radiant energy and strip electrons from water, producing molecular oxygen as a byproduct (1, 2). Highly reactive forms of oxygen are also generated, and despite the presence of detoxifying enzymes, photooxidative damage is a serious problem. Although there has been extensive study of the damage, protection, and repair of photosynthetic membranes, there has been little attention paid to the genetic consequences of photooxidative stress (3). This can be attributed, at least in part, to a grossly incomplete knowledge of how cpDNA is replicated and maintained throughout the life of a plant (3, 4).However, despite the high-stress environment, the evolution of cpDNA is more conservative than nuclear or mitochondrial DNA (5). We can thus infer that DNA repair mechanisms play important roles in protecting cpDNA and are intimately involved in its evolution. And although some processes have been identified (6-11), the panoply of organelle repair mechanisms is not known, nor is it clear which are most important, or how they have coevolved with the genome. Although we expect that some cpDNA repair processes will reflect its prokaryotic ancestry, the consequences of eukaryotic cell integration...
