Book of Abstracts: Albany 2005
Isolation and Characterization of the Proteins Recognizing Oxidatively Damaged DNA
Oxidizing agents and ionizing radiation damage DNA by inducing a variety of lesions, including nicks and gaps in one strand of a DNA duplex (1), as well as base damage. How cells repair such lesions has attracted the attention of many researchers. At present, the damage-sensing step is still not well understood for oxidative lesions in the DNA backbone (2). Previous work in our laboratory revealed the chemical nature of the nicks and gaps that are introduced into the DNA backbone by the hydroxyl radical (3, 4), which is the source of the oxidative damage that is produced by ionizing radiation. Other experiments suggested that hydroxyl radical-induced gapped DNA may have a distinctive bent structure (5). It has been reported that, in some systems, nicked and gapped DNA are strongly bent when bound to repair proteins (6, 7). Thus, we proposed (5) that this distinctive structural feature may be recognized by the proteins that initiate the oxidative damage repair pathway.
We made use of our knowledge of the structures of oxidative lesions to design a DNA oligonucleotide duplex that has a specific backbone gap. This oligonucleotide was then used as ?bait? to catch yeast proteins that bind specifically to this lesion. For our experiments, we chemically synthesized a gapped DNA oligonucleotide duplex. We obtained protein extract from a yeast culture and fractionated it by gel filtration. The Electrophoretic Mobility Shift Assay (EMSA) was used to detect specific binding of proteins from the yeast extract to gapped DNA. We plan to use mass spectrometry to characterize these proteins. Our results will contribute to the understanding of the early stages of repair of oxidative lesions in DNA.
This research was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R01 GM41930).
References and Footnotes
Department of Chemistry