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Kinetic studies of human tyrosyl-DNA phosphodiesterase, an enzyme in the topoisomerase I DNA repair pathway.

Abstract
Tyrosyl-DNA phosphodiesterase (TDP) cleaves the phosphodiester bond linking the active site tyrosine residue of topoisomerase I with the 3' terminus of DNA in topoisomerase I-DNA complexes which accumulate during treatment of cancer with camptothecin. In yeast, TDP mutation confers a 1000-fold hypersensitivity to camptothecin in the presence of an additional mutation of RAD9 gene [Pouliot, J.J., Yao, K.C., Robertson, C.A. & Nash, H.A. (1999) Science 286, 552-555]. Based on the recently solved crystal structure, human TDP belongs to a distinct class within the phospholipase D superfamily in spite of very low sequence homology [Interthal, H., Pouliot, J.J. & Champoux, J.J. (2001) Proc. Natl Acad. Sci. USA 98, 12009-12014, and Davies, D.R., Interthal, H., Champoux, J.J. & Hol, W.G.J. (2002) Structure 10, 237-248]. To understand the enzymatic mechanism of this novel enzyme, and to facilitate inhibitor screening of human TDP, we have expressed and purified recombinant human TDP variants carrying deletions of 1-39 or 1-174 amino acids. Furthermore, a continuous colorimetric assay in a 96-well format was also developed using p-nitrophenyl-thymidine-3'-phosphate as substrate. This assay system is able to detect enzymatic activity at enzyme concentrations as low as 15 nm. Purified recombinant human TDPNDelta39 cleaved p-nitrophenyl-thymidine-3'-phosphate with Km and kcat values of 211.14 +/- 23.83 micro m and 8.82 +/- 0.57 per min in the presence of Mn2+.
AuthorsTing-Jen Cheng, Peter G Rey, Thomas Poon, Chen-Chen Kan
JournalEuropean journal of biochemistry (Eur J Biochem) Vol. 269 Issue 15 Pg. 3697-704 (Aug 2002) ISSN: 0014-2956 [Print] England
PMID12153566 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
Chemical References
  • Recombinant Proteins
  • thymidine 5'-4-nitrophenyl phosphate
  • Thymidine Monophosphate
  • Phosphoric Diester Hydrolases
  • TDP1 protein, human
  • tyrosyl-DNA phosphodiesterase
Topics
  • Amino Acid Sequence
  • Colorimetry (methods)
  • Escherichia coli (genetics)
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Phosphoric Diester Hydrolases (chemistry, genetics, metabolism)
  • Recombinant Proteins (chemistry, genetics, metabolism)
  • Sensitivity and Specificity
  • Sequence Homology, Amino Acid
  • Thymidine Monophosphate (analogs & derivatives, metabolism)
  • Yeasts (enzymology)

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