| Oxidative damage to DNA has attracted intense research interest with considerable attention focused on base lesions. More than 20 DNA base lesions have been identified, and mechanisms for their repair elucidated. However, for oxidative lesions in the DNA backbone, the nature of the damage-sensing step in their cellular repair remains unclear.;In this dissertation, I examined a synthesized, gapped DNA probe as a model system. The structure of the lesion in the gapped DNA probe corresponds to one type of break found in oxidatively damaged DNA produced by the hydroxyl radical. Electrophoretic Mobility Shift Assay (EMSA) experiments demonstrated an appreciable shift due to the specific binding of yeast proteins to gapped DNA. To isolate the specific binding proteins, I subjected yeast protein extract to fractionation, EMSA gel purification and electroelution, and then electrophoresed the electroeluted proteins on an SDS gel. I then excised the candidate protein bands to prepare samples for mass spectrometry analysis to initially characterize the gapped DNA-binding protein.;The second part of the dissertation presents a new protocol for next-generation hydroxyl radical footprinting that incorporates the Solexa sequencing platform. Emerging next-generation DNA sequencing technologies work up to 200 times faster and cheaper than the conventional Sanger method, and have already brought profound changes to genomic research. In my protocol, I used the Solexa platform, one of the next-generation sequencing technologies, to map hydroxyl radical cleavage patterns in genomic DNA. To meet the Solexa platform's sample requirements, fragmented DNA must fall in a size range of 100-200 bp. Optimization of the conditions of hydroxyl radical cleavage can achieve this objective. Then I used T7 endonuclease I to convert gap sites into double-stranded breaks. Solexa sequencing of DNA samples produced with my protocol agrees well with the theoretically predicted cleavage pattern. The new method will enable researchers to expand the application of hydroxyl radical footprinting to the genome-wide scale with high efficiency. |
