Determination of DNA Lesions Gel Electrophoresis Method

1. Leaf tissue processing: Unless otherwise noted, all procedures were performed under dim yellow light (General Electric, fluorescent; > ~500 nm) to reduce the possibility of undesired photoreactivation of the samples during processing. Leaf tissue was processed according to the methods outlined by several researchers (Quaite et al., 1992; Quaite et al., 1994; Sutherland et al., 1996; Kang et al., 1998;

Bennett et al., 2001). Leaf disks were chopped and ground to a fine, light green powder in liquid nitrogen (LN2), dropped into Lysis Buffer #1 (10 mM Tris-HCl, pH 8.0, 0.83 M EDTA, 13% mannitol, 2% sarcosyl, and 1 mg/ml Proteinase K (Boehringer Mannheim, Indianapolis, IN), 40°C -45°C) and vacuum-infused. Agarose Sea Plaque (2-1/2% w/v, FMC, Rockland, ME) in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, heated to 70°C - 75°C) was added and the mixture pipetted into chilled molds (GeNunc Module, Nalge Nunc, catalog no. 2-32549). The solidified samples were covered with Lysis buffer #2 (10 mM Tris-HCl pH 8.0, 0.83 M EDTA, 2% sarcosyl, and 1 mg/ml Proteinase K), wrapped with aluminum foil, and incubated 24 hours at 45 °C. Lysis buffer #2 was replaced with Lysis buffer #3 (10 mM Tris-HCl pH 7.8 - 8.0, 0.1 M EDTA, 20 mM NaCl, 1% sarcosyl, and 1 mg/ml Proteinase K), rewrapped in foil, and incubated for 24 to 48 hours at 45°C. Lysis buffer #3 was refreshed, and the plugs incubated for an additional 24 to 48 hours at 45°C (total of 72-hour minimum in Lysis buffer #3).

2. Plug processing: Plugs were soaked in TE (pH 7.6) in the dark and over ice. Plugs were subsequently soaked in TE and phenylmethyl sulfonyl fluoride (PMSF) (2.2 ^L/ml) 2X at room temperature, with a final cold TE rinse. Samples were soaked 3X in UV endonuclease buffer (30 mM Tris-HCl, pH 7.6, 40 mM NaCl, 1 mM EDTA) over ice. Two similar plugs were placed in a single labeled Eppendorf centrifuge tube, placed in a water bath (70°C - 72°C) and the re-melted DNAagarose mixture pipetted (7 |iL) to form new plugs. The new plugs were covered with UV endonuclease buffer and stored overnight (4°C). The buffer was replaced with 20 ^L Reaction Mix (UV endonuclease buffer containing 1 mM dithiothreitol (DTT) and 0.1 mg ml-1 bovine serum albumin (BSA)), and the samples were kept on ice.

3. Plug preparation: Plugs were divided into two groups: the "Minus" plugs and the "Plus" plugs. The Reaction Mix was refreshed in the "Minus" plugs, removed from the "Plus" plugs, and then replaced with a solution of Reaction Mix and sufficient Micrococcus luteus UV endonuclease (MLE) enzyme to cut the DNA at all dimer locations. The preparation of MLE used in these experiments cleaved approximately 4 x 1015 CPDs ^L-1h-1. The relative numbers of DNA fragments in each "Plus" plug (sample cut with MLE) and "Minus" plug (sample not exposed to the enzyme) were determined. All plugs were kept on ice for 30 minutes and then incubated at 37°C for 60 minutes. Additional Reaction Mix (2 ^L) was added to the "Minus" plugs, and an equal volume of Reaction Mix with 50% MLE was added to the "Plus" plugs. Samples were incubated at 37°C for 30 minutes and held on ice until processed. All plugs were rinsed 2X with TE, covered with alkaline stop (0.5 M NaOH, 50% (vol/vol) glycerol and 0.25% (wt/vol) bromocresol green), and stored on ice at 4°C. The gels (agarose (SeaKem LE; FMC, Rockland, ME) (0.4% wt/vol) in 1 mM EDTA, 50 mM NaCl) were prepared and equilibrated with alkaline electrophoresis solution (2 mM EDTA, 30 mM NaOH), at room temperature. The molecular length standards from bacteriophages

G (750kb), T4 (170 kb), 2 (48.5 kb), the Hindlll digest of 2 (23, 9.4, 6.6, 4.3, 2.1 kb) and BioLow (1.0, 0.5, and 0.2 kb) (BioMarker Low, BioVentures, Inc., Murfreesboro, TN) were prepared, and treated with alkaline stop for 60 minutes. All sample plugs were placed in a water bath (at 37°C) for 60 minutes to denature the DNA, rinsed with cold alkaline electrophoresis solution (30 mM NaOH, 2 mM EDTA), and the companion pair of "Plus" and "Minus" samples were loaded into adjacent gel wells.

4. Static field electrophoresis: The gel rig was placed in a 10°C water bath to provide temperature stability. The gel was subjected to a static field electrophoresis for 15 minutes at 3 volts/cm. Recirculation of the electrophoresis solution was begun, and the gel electrophoresed for an additional 25 minutes at 3 volts/cm.

5. Unidirectional pulsed-field electrophoresis (UPFE): This method improves the resolution of DNA molecules longer than 100 kilobases (kb), and increases the ability to measure low frequencies of lesions (Sutherland et al., 1987; Quaite et al., 1992; Quaite et al., 1994) that can occur in DNA exposed to ultraviolet light. Electrophoresis resumed with UPFE (15 volts/cm; 0.3 second pulse with 10 second interpulse period, 10°C with buffer recirculation) for 7 hours ± 1 hour. The DNA plugs were removed, and the electrophoresis continued for a total of 24 hours. The DNA fragments were dispersed in the gel according to their singlestrand molecular lengths. The gel was rinsed, neutralized 2X with 0.1 M Tris-HCl pH 8.0, and stained with ethidium bromide (0.67 ^g/mL). Gels were stored at 4°C until imaged.

6. Electronic imaging: A quantitative (digital) electronic image was obtained using a charge-coupled device-based (CCD) system designed and built by J. C. Sutherland (Sutherland et al., 1987). The dual-camera system gave a linear response to the DNA-bound ethidium fluorescence. By obtaining the optical density profile in digital form of the DNA in each lane within the gel, median migration distances were calculated by the computer (Sutherland et al., 1990). The distribution of fluorescence was measured. Results were proportional to the quantity of DNA at each position within the lane (Sutherland et al., 1990).

7. Analysis and dimer quantification: The number of breaks in the DNA molecule was derived from the total number of DNA molecules in the MLE treated sample, less the number of molecules in the untreated sample. In general, molecules in the "Plus" sample are shorter than those in the "Minus" sample (Freeman et al., 1986). The standard sensitivity using this method is approximately 1 lesion/Mb, with a current limit sensitivity of about 1 lesion/5 Mb. In a preliminary action spectrum using very high irradiance levels (0 kW m - 15 kW m ), nearly 120 dimers were measured (data not shown), showing that these isolines are capable of accumulating a very large amount of DNA damage (Pope, 2001). Samples containing a known quantity of dimers (~30 CPDs - 60 CPDs) were periodically run with the test samples to verify the analytic procedures, providing a positive control.

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