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

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, 1200912014, and Davies, D.R., Interthal, H., Champoux, J.J. & Hol, W.G.J. (2002) Structure 10, 237-2481. 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 rim. Purified recombinant human TDPNDelta39 cleaved p-nitrophenyl-thyniidine-3'-phosphate with K-m and k(cat) values of 211.14 +/- 23.83 muM and 8.82 +/- 0.57 per min in the presence of Mn2+.