Evidence for base excision repair of oxidative DNA damage in chloroplasts of Arabidopsis thaliana.
Chloroplasts are the sites of photosynthesis in plants, and they contain their own multicopy, requisite genome. Chloroplasts are also major sites for production of reactive oxygen species, which can damage essential components of the chloroplast, including the chloroplast genome. Compared with mitochondria in animals, relatively little is known about the potential to repair oxidative DNA damage in chloroplasts. Here we provide evidence of DNA glycosylase-lyase/endonuclease activity involved in base excision repair of oxidized pyrimidines in chloroplast protein extracts of Arabidopsis thaliana. Three base excision repair components (two endonuclease III homologs and an apurinic/apyrimidinic endonuclease) that might account for this activity were identified by bioinformatics. Transient expression of protein-green fluorescent protein fusions showed that all three are targeted to the chloroplast and co-localized with chloroplast DNA in nucleoids. The glycosylase-lyase/endonuclease activity of one of the endonuclease III homologs, 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 third glycosylase activity and potentially another pathway for repair of oxidative DNA damage in chloroplasts.
Gutman
Benjamin L
BL
From the Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102.
Niyogi
Krishna K
KK
From the Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102. Electronic address: niyogi@nature.berkeley.edu.
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D018742:DNA, Chloroplast / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D017343:Genes, Plant; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D016254:Mutagenesis, Insertional; D009154:Mutation; D030821:Plants, Genetically Modified; D017382:Reactive Oxygen Species / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D018742:DNA, Chloroplast; D017382:Reactive Oxygen Species; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C540283:Arp protein, Arabidopsis; D004720:Endonucleases; D045643:DNA Repair Enzymes