RESULTS Effect of UV on the Turnover of Chloroplast

A UV-specific endonuclease was used to detect ultraviolet light-induced pyrimidine dimers in chloroplast DNA of Chlamydomonas veinhavdi that was specifically labeled with tritiated thymidine. All of the dimers induced by 100 J/m of 254 nm light are removed by photoreaction. Wild-type cells exposed to 50 J/m of UV light removed over 80% of the dimers from chloroplast DNA after 24 h of incubation in growth medium in the dark. A UVsensitive mutant, UVS 1, defective in the excision of pyrimidine dimers from nuclear DNA is capable of removing pyrimidine dimers from chloroplast DNA nearly as well as wild-type, suggesting that nuclear and chloroplast DNA dark-repair systems are under separate genetic control. INTRODUCTION The presence and biological importance of DNA in organelles of eukaryotic cells has been well documented . The existence in practically all cells of mechanisms for the repair of damaged DNA is also well established. Three DNA repair systems for coping with ultraviolet light-induced pyrimidine dimers have been elucidated primarily from work with bacteria; these are (1) photoreactivation, (2) excision-repair, and (3) postreplication repair. Clayton, p Doda and Friedberg failed to find evidence for any of these repair mechanisms operating on pyrimidine dimers induced in mitochondrial DMA of mammalian cells. Pyrimidine dimers can be removed from yeast mitochondrial DNA by 3 4 photoreactivation although excision-repair is apparently absent ' . The only previous study of chloroplast DNA repair failed to find evidence for excision of dimers or for repair replication in Chlamydomonas chloroplasts. However, these workers also reported a lack of excision of pyrimidine dimers from nuclear DNA of Chlamydomonas. Using a more sensitive assay for pyrimidine dimers, thus permitting a smaller fluence of irradiation, we found that Chlamydomonas does remove pyrimidine dimers from nuclear DNA in the dark . We have extended the study of DNA repair in Cklamydomonas to chloroplast DNA. We find evidence for repair of pyrimidine dimers in chloroplast DNA both by photoreactivation and by a dark-repair process which may be excision-repair. 2893 © Information Retrieval Limited 1 Falconberg Court London W 1 V 5 F G England Nucleic Acids Research MATERIALS AND METHODS Chlamydomonas strains—Chlamydomonas strains used have been described in a previous publication . Detection of pyrimidine dimers in chloroplast DNA—Chlamydomonas was grown in TAP medium in the presence of 10 viCi/ml of [methyl-H]-thymidine (50 Ci/mmole, New England Nuclear or ICN) for two days to a final cell density of 5 x 10 to 1 x 10 cells/ml. The cells were washed and resuspended in fresh TAP medium to a cell density of 1 x 10 cells/ml. Thirteen to 15 ml were irradiated in a 15 cm petri dish under a germicidal lamp at a fluence rate of 5 J/m per second. (It should be noted that fluences given in the prior publication must be multiplied by a factor of 2 for proper comparison to this paper due to a faulty UV meter.) The cells were diluted to 5 x 10 cells/ml with TAP medium before incubation in the dark at 26 C with shaking. The extraction, treatment of the isolated DNA with the UVspecific endonuclease, and sedimentation through alkaline sucrose gradients were described in an earlier publication . Centrifugation was for 210 minutes at 30,000 rpm with a SW 50.1 rotor at 20 C except where noted otherwise. Bacteriophage T7 DNA was used as a molecular weight marker. Measurement of turnover of chloroplast DNA—Chlamydomonas was grown as described above except 0.06 to 0.2 yCi/ml of [C] adenine (53.5 mCi/mmole) was present in addition to 10 yCi/ml of [H] thymidine. Adenine labels both Q nuclear and chloroplast DNA whereas thymidine is incorporated specifically into chloroplast DNA . Irradiation, incubation in the dark and extraction of DNA were the same as described above. Nuclear and chloroplast DNA were resolved on CsCl density gradients performed as previously described . Turnover of chloroplast DNA was calculated from the ratio of tritium in the chloroplast DNA to '"C in the nuclear DNA. Determination of chlovoplast DNA synthesis after UV-irradiation— Chlamydomonas DNA was prelabeled by growing in the presence of 0.06 to 0.2 yCi/ml [^C] adenine to a cell density of 5 x 10 to 1 x 10 cells/ml. Irradiation was done as described previously. The irradiated cells, along with unirradiated controls, were incubated in the dark at 26 C with shaking for 24 h in TAP medium in the presence of 16 to 20 uCi/ml of £H] thymidine. The DNA was extracted and the nuclear and chloroplast DNA separated on CsCl density gradients. The amount of chloroplast DNA synthesized was estimated from the ratio of tritium in chloroplast DNA to C in nuclear DNA.