Book of Abstracts: Albany 2005
Probing the Sequence-dependent Structure of DNA with the Hydroxyl Radical
It has become apparent that the local structure of DNA significantly deviates from the canonical B-form throughout the genome. Unusual DNA structures are involved in many critical cellular processes including transcription and replication. As the local structure of DNA is sequence-dependent (1, 2), it is likely that local structural features, rather than solely the sequence of nucleotides, are important for site-specific protein recognition. Current genome projects are aimed at solving only the sequence of the genome. While the data obtained will no doubt prove invaluable, this represents only a one-dimensional dataset. In order to gather a more complete understanding of the workings of the genome, a structural map would be helpful.
The purpose of this project is twofold: i) to devise a method for producing detailed structural maps of genomic DNA and ii) to use the structural data along with the sequence data to find unknown protein binding sites as well as other unusual structural features. For this purpose, the hydroxyl radical has been employed. DNA is cleaved by the hydroxyl radical at every nucleotide, in a controlled fashion, and the extent of cutting is quantified by denaturing gel electrophoresis. The resulting cleavage pattern is reproducible and closely related to details of the local three-dimensional structure of the molecule (3, 4).
Several hundreds of random sequence cleavage patterns have been collected and organized into a database for analysis. Software is currently under development to determine the relationship between the sequence and structure of DNA, as well as to identify unknown protein binding sites by analysis of these patterns. Our methods of cleavage pattern prediction have proven to be accurate, and our application of these data to biological problems has shown great potential.
References and Footnotes
Jason A. Greenbaum1