Published online 27 August 2004
Nucleic Acids Research, Vol. 32 No. 15 © Oxford University Press 2004; all rights reserved
Design and synthesis of fluorescent substrates for human tyrosyl-DNA phosphodiesterase I
1 deCODE biostructures, 7869 NE Day Road West, Bainbridge Island, WA 98110, USA and 2 San Diego State University, Biology Department, 5500 Campanile Drive, San Diego, CA 98182-4614, USA
* To whom correspondence should be addressed. Tel: +1 206 780 8535; Fax: +1 206 780 8549; Email: aburgin{at}decode.com
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received June 30, 2004; Revised and Accepted August 10, 2004
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a DNA repair enzyme that acts upon protein–DNA covalent complexes. Tdp1 hydrolyzes 3'-phosphotyrosyl bonds to generate 3'-phosphate DNA and free tyrosine in vitro. Mutations in Tdp1 have been linked to patients with spinocerebellar ataxia, and over-expression of Tdp1 results in resistance to known anti-cancer compounds. Tdp1 has been shown to be involved in double-strand break repair in yeast, and Tdp1 has also been implicated in single-strand break repair in mammalian cells. Despite the biological importance of this enzyme and the possibility that Tdp1 may be a molecular target for new anti-cancer drugs, there are very few assays available for screening inhibitor libraries or for characterizing Tdp1 function, especially under pre-steady-state conditions. Here, we report the design and synthesis of a fluorescence-based assay using oligonucleotide and nucleotide substrates containing 3'-(4-methylumbelliferone)-phosphate. These substrates are efficiently cleaved by Tdp1, generating the fluorescent 4-methylumbelliferone reporter molecule. The kinetic characteristics determined for Tdp1 using this assay are in agreement with the previously published values, and this fluorescence-based assay is validated using the standard gel-based methods. This sensitive assay is ideal for kinetic analysis of Tdp1 function and for high-throughput screening of Tdp1 inhibitory molecules.
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