DNA repair in the chloroplast

By necessity of their photoautotrophy and their immobility plants both require light and cannot avoid it. While providing energy via photosynthesis, light also causes photooxidative damage. The chloroplast is the organelle that absorbs and uses light energy in photosynthesis. Thus it is the organelle most likely to take the brunt of photooxidation. Chloroplasts, being of endosymbiotic origin, contain their own self-replicating, multicopy requisite genome (plastome). A high potential exists for oxidative damage to the plastome. To identify and begin to understand the mechanisms of DNA repair in the chloroplast, I have taken three independent, but complementary approaches.; Using a biochemical approach, I demonstrated that there is DNA glycosylase/lyase activity in chloroplast extracts of Arabidopsis thaliana, showing for the first time that such an activity exists in chloroplasts. Three potentially overlapping components of base excision repair (BER), two Nth homologs and an AP endonuclease that might account for this activity were identified by bioinformatics. Transient expression of protein-GFP fusions showed that all three are targeted to the chloroplast and localized in chloroplast nucleoids. The glycosylase/lyase activity of one Nth homolog (AtNTH2), which had not previously been characterized, was confirmed in vitro. T-DNA insertions in each of these genes were identified, and the physiological and biochemical phenotypes of the single, double and triple mutants were analyzed. This mutant analysis revealed the presence of a new glycosylase activity and potentially another DNA-repair pathway for addressing this type of oxidative damage (oxidized pyrimidines).; In an expansion of the reverse-genetics approach, eight additional Arabidopsis candidate chloroplast DNA-repair genes, were identified bioinformatically. Analysis of the genes and corresponding T-DNA mutants eliminated two as encoding nonchloroplastic proteins, and confirmed another as chloroplastic.; In a third approach, I analyzed some aspects of the plastome mutation rate in Chlamydomonas reinhardtii. Specifically, I more closely analyzed a mutant, npq1 lor 1, deficient in chloroplast xanthophylls to test whether its increased photooxidative stress affected the mutation rate in the chloroplast. Subsequently I developed a novel Chlamydomonas reporter strain for a chloroplast mutator screen. The strain was designed to indicate oxidative DNA mutations by activation of a chloroplast reporter construct. I screened several UV mutageneses on this strain.