Braking the Double Helix: Effects of UV Radiation on Super-Coiled DNA

Overview

Objectives/Goals UV radiation has enough energy to nick and break the phosphate backbone of DNA. Last year I proved that natural levels of solar UV radiation were sufficient to nick or break the DNA. This year I asked if the breakage was occuring randomly or at specific sites. Methods/Materials I used a super-coiled plasmid for this project because, on an agarose gel, the broken parts of the DNA move at different speeds. The band that has moved the farthest is the super-coiled DNA, in the middle is the linear DNA (broken), and the band that has moved the least, is the open circle DNA (nicked). To identify the super-coiled band on the gel, I performed an Ethidium Bromide (EtBr) test. In this test I added a range of EtBr concentrations (for a final concentration of 0.1 µg/mL to 5 mg/mL) to the super-coiled DNA. As the EtBr intercalates into the DNA, the plasmid is forced into an open-circle. As more EtBr is added, the plasmid is forced to positively super-coil. To find the linear DNA, I used DNA digested with EcoR1. After adding the EtBr to the DNA, I let it sit for 15 minutes at room temperature then loaded it into the gel. I ran it as described under Gel Electrophoresis. To test the sequence specificity of the breakage, I took, pUC19, and put it under a UV sterilization lamp. Replicate DNA samples were exposed for up to 1 hr. I digested the exposed DNA with three restriction enzymes, Bam H1, EcoR1, and Hind III and then ran the digested DNA on a 1.2% agarose gel. Results I found distinct bands on the gel, below the super-coiled (migrated faster), which suggests that some parts of the backbone are more susceptible to breakage than others. I was surprised to find that in the digested DNA, some open circle remained, increasing in amount with the amount of UV exposure. I interpreted that as the enzyme reached the nick in the DNA, and broke off because it was unable to go beyond the nick. Conclusions/Discussion I concluded that the DNA is more susceptible to brakage on the phosphate backbone.

Competition history

• CSEF 2008 Biochemistry/ Molecular Biology · Entry S0416

Resources

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