Construction of a database of hydroxyl radical DNA cleavage data to determine the sequence dependence of DNA structure

The sequence-dependence of DNA structure plays an important biological role in gene regulation and expression. Over the past 20 years this problem has attracted numerous studies involving many different techniques, however, none of these studies so far has been able to derive a general model to explain how the sequence of DNA governs its local structure. In this dissertation, I describe the use of the hydroxyl radical cleavage technique to address this problem. The hydroxyl radical cleavage pattern of a DNA molecule directly correlates with the local three-dimensional structure of the double helix. By collecting hundreds of hydroxyl radical cleavage patterns from different DNA sequences, I have been able to construct a DNA sequence-dependent structural database.;The hydroxyl radical cleavage experiment was first performed using the conventional approach involving radiolabeled DNA. Although this method was shown to generate useful results, it was not suitable for the acquisition of an extensive dataset due to the tedious nature of the experimental procedure. After solving many technical problems, I developed a new, high-throughput, automated fluorescence-based protocol for the hydroxyl radical cleavage experiment. After careful calibration, the new system provided a remarkable improvement in efficiency: complete data acquisition times were reduced from 150 hr to 15 hr. Results derived from this system demonstrated excellent accuracy and consistency.;Hydroxyl radical cleavage reactions on more than 80 different DNA sequences provided a large number of cleavage patterns which have been assembled into a relational database. Using this database, we have formulated a trinucleotide sequence-dependent structural model for DNA, which allows us to predict the hydroxyl radical cleavage pattern of any DNA sequence. This model has been used to search for transcription factor binding sites in genomic DNA. The extensive collection of DNA structural data that can now be obtained from the hydroxyl radical cleavage experiments using my protocol will enable researchers to make a detailed structural map of genomic DNA, and to develop higher-level models of DNA structure, with more powerful capabilities for structure prediction.