Book of Abstracts: Albany 2007
June 19-23 2007
Isolation of Proteins Involved in Recognition of Oxidatively Damaged DNA from the Yeast Saccaromyces cerevisiae
Our group has been studying the structures and properties of the nicked and gapped DNA molecules that are the product of oxidative damage caused by the hydroxyl radical (1, 2). This work has enhanced understanding of the chemical nature of oxidative lesions in the DNA backbone, while fostering our interest in how oxidatively damaged DNA is repaired by the cell. It remains unclear as to the nature of the damage-sensing step in cellular repair of oxidative lesions in the DNA backbone (3).
Previous work from our lab showed that gapped DNA produced by hydroxyl radical treatment has a distinctive bent structure (4). Bent DNA molecules are found in three-dimensional structures of nicked and gapped DNA molecules bound to repair proteins (5, 6) in some systems. We proposed(4) that this distinctive structural feature may be recognized by proteins that initiate the process of oxidative damage repair.
To discover which cellular proteins bind to and recognize oxidatively-damaged DNA, we synthesized a gapped DNA oligonucleotide duplex. The gapped DNA molecule was used as "bait" to attract yeast proteins that recognize this lesion. A yeast protein extract was fractionated and then incubated with gapped DNA. The Electrophoretic Mobility Shift Assay (EMSA) was used to detect specific binding. Proteins in specific binding bands were recovered from the EMSA gel and then electrophoresed and visualized on an SDS gel. Mass spectrometry was employed to identify the proteins.
This research was supported by the National Institutes of Health (R01 GM41930).
References and Footnotes
Long Xu* and Thomas D. Tullius
Department of Chemistry,