Repair of damaged DNA by Arabidopsis cell extract.
All living organisms have to protect the integrity of their genomes from a wide range of genotoxic stresses to which they are inevitably exposed. However, understanding of DNA repair in plants lags far behind such knowledge in bacteria, yeast, and mammals, partially as a result of the absence of efficient in vitro systems. Here, we report the experimental setup for an Arabidopsis in vitro repair synthesis assay. The repair of plasmid DNA treated with three different DNA-damaging agents, UV light, cisplatin, and methylene blue, after incubation with whole-cell extract was monitored. To validate the reliability of our assay, we analyzed the repair proficiency of plants depleted in AtRAD1 activity. The reduced repair of UV light- and cisplatin-damaged DNA confirmed the deficiency of these plants in nucleotide excision repair. Decreased repair of methylene blue-induced oxidative lesions, which are believed to be processed by the base excision repair machinery in mammalian cells, may indicate a possible involvement of AtRAD1 in the repair of oxidative damage. Differences in sensitivity to DNA polymerase inhibitors (aphidicolin and dideoxy TTP) between plant and human cell extracts were observed with this assay.
Li
Anatoliy
A
Institute of Plant Sciences, Eidgenössische Technishe Hochschule, Universitätsstr 2, CH-8092 Zürich, Switzerland.
Schuermann
David
D
Gallego
Francesca
F
Kovalchuk
Igor
I
Tinland
Bruno
B
D016590:Aphidicolin / Q000494:pharmacology; D017360:Arabidopsis / Q000166:cytology / Q000235:genetics* / Q000528:radiation effects; D002457:Cell Extracts; D002945:Cisplatin / Q000494:pharmacology; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D004260:DNA Repair / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D004268:DNA-Binding Proteins; D054306:Dideoxynucleotides; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D018745:Genome, Plant; D006801:Humans; D008751:Methylene Blue / Q000494:pharmacology; D019384:Nucleic Acid Synthesis Inhibitors; D018384:Oxidative Stress; D030821:Plants, Genetically Modified; D013942:Thymine Nucleotides / Q000494:pharmacology; D014466:Ultraviolet Rays
C094077:3'-nitro-2',3'-dideoxythymidine 5'-triphosphate; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D054306:Dideoxynucleotides; D019384:Nucleic Acid Synthesis Inhibitors; D013942:Thymine Nucleotides; D016590:Aphidicolin; D004720:Endonucleases; D002945:Cisplatin; D008751:Methylene Blue