DOUBLE-STRAND BREAK-INDUCED RECOMBINATION IN CHLAMYDOMONAS-REINHARDTII CHLOROPLASTS

The mechanisms of chloroplast recombination are largely unknown, Using the chloroplast-encoded homing endonuclease I-Crel from Chlamydomonas reinhardtii, an experimental system is described that allows the stud y of double strand break(DSB)-induced recombination in chloroplasts, T he I-Crel endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtii and cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombinatio n in chloroplast DNA, the genes encoding the I-Crel endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S ge ne, was integrated at an ectopic location, Endonuclease function was t ransiently provided by mating the strains containing the recombination substrate to a wild-type strain, The outcome of DSB repair was analyz ed in haploid progeny of these crosses, Interestingly, resolution of D SB repair strictly depended upon the relative orientation of the ectop ic ribosomal cDNA and the adjacent copy of the 23S gene, Gene conversi on was observed when the 23S cDNA and the neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the int ron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in direc t repeat orientation relative to the proximal 23S gene resulted in a d eletion between the 23S cDNA and the 23S gene, These results demonstra te that C.reinhardtii chloroplasts have an efficient system for DSB re pair and that homologous recombination is strongly stimulated by DSBs in chloroplast DNA.