CHED 360 |
| A non-radioactive assay for detection of DNA-protein crosslinking, based on inhibition of enzymatic cleavage of DNA, was developed to determine the susceptibility of specific DNA sites to 1-electron oxidation and oxidative crosslinking. Guanine is the most easily oxidized DNA base, and its oxidation potential decreases in the order: -GGG- > -GG- > -GX- where X is not G. Therefore inhibition of enzymatic cleavage should be observed if crosslinks occur at those sites. Oxidation of guanine sites in linearized pBR322 DNA was accomplished by the flash-quench technique. The inhibition of cleavage from BamHI (5'-G/GATCC-3') and EcoRI (5'-G/AATTC-3') by DNA-protein crosslinks was observed by agarose gel electrophoresis, confirming DNA-protein crosslinks at guanine. In contrast, it was found that the enzyme, AseI (5'-AT/TAAT-3'), which cleaves an AT sequence, was not inhibited. These results show that sites of oxidative DNA damage can be mapped out using restriction enzymes. |
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Undergraduate Research Poster Session: Analytical Chemistry
11:00 AM-1:00 PM, Monday, March 26, 2007 Hyatt Regency Chicago -- Riverside Center, Poster
Division of Chemical Education |