Repair of damaged DNA by Arabidopsis cell extract

Abstract

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.

Figure 1.

Scheme of the in Vitro Repair Assay for Arabidopsis Whole-Cell Extract. For a detailed description of the each step, see Methods.

Figure 2.

In Vitro Repair Synthesis of Damaged Plasmid DNA in Arabidopsis Cell Extract. SmaI-digested pGEX-3X and pBSK plasmid DNA were damaged as indicated and incubated with a plant extract in the presence of DIG-labeled dUTP. After purification, plasmid DNA was separated by agarose gel electrophoresis, transferred to a membrane, and detected with anti-DIG–alkaline phosphatase antibodies. Chemiluminescence was registered on x-ray film. For both (A) and (B), gel at top and film at bottom. (A) Lane 1, nondamaged pGEX-3X plus pBSK; lane 2, UV light–irradiated pGEX-3X; lane 3, nondamaged pBSK; lane 4, UV light–irradiated pGEX-3X plus nondamaged pBSK; lane 5, nondamaged pGEX-3X plus UV light–irradiated pBSK. (B) Nondamaged pBSK was used as an internal control, and pGEX-3X was treated as follows: lane 1, UV light irradiation (450 J/m²); lane 2, 10 μg/mL methylene blue and 30 min of illumination by visible light; lane 3, 10 μg/mL methylene blue and 60 min of illumination by visible light; lane 4, 1 μM cisplatin; lane 5, 5 μM cisplatin; lane 6, 10 μM cisplatin.

Figure 3.

Inhibition of luciferase Expression by Different DNA-Damaging Agents. pPP22 plasmid DNA containing a firefly luciferase gene was damaged as indicated by UV light (A), 10 μg/mL methylene blue and visible light (B), or cisplatin (C) and used to transform tobacco protoplasts. pRR22 plasmid DNA expressing Renilla luciferase was cotransformed as an internal control. After recovery, the activity of firefly luciferase was measured and standardized against Renilla luciferase activity. The relative firefly luciferase activity of damaged plasmid DNA was compared with that of nondamaged DNA (100% activity). Error bars represent standard deviations of the means of two independent experiments.

Figure 4.

In Vitro Repair Synthesis in Cell Extracts from Wild-Type and AtRAD1 Antisense Plants. Standard repair reactions were performed on pGEX-3X DNA damaged by 450 J/m² UV light, by 10 μg/mL methylene blue and 45 min of illumination with visible light, and by 5 μM cisplatin. Nondamaged pBSK was used as an internal control. A2, A5, and A12, antisense plants; WT, wild type.

Figure 5.

Inhibition of Plant Growth by H₂O₂ and Cisplatin. Wild-type (WT) and AtRAD1 antisense (A2, A5, and A12) plants were grown on liquid medium containing the indicated concentrations of H₂O₂ (A) and cisplatin (B). After 10 days, plants were harvested and the weight of each individual plant was measured. Error bars represent standard deviations from 12 independent plants. Asterisks indicate values that are not statistically different from those of the wild type (P > 0.05) using Student's t test.

Figure 6.

Effect of DNA Polymerase Inhibitors on in Vitro Repair Synthesis. (A) HeLa cell extract. (B) Arabidopsis cell extract. Standard repair reaction was performed on pGEX-3X DNA damaged by UV light (lanes 1 to 3), methylene blue (lanes 4 to 6), and cisplatin (lanes 7 to 9). Aphidicolin (100 μM) and ddTTP (100 μM) were added where indicated. Nondamaged pBSK DNA was used as a control template.