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, 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 TDPNΔ39 cleaved p‐nitrophenyl‐thymidine‐3′‐phosphate with Km and kcat values of 211.14 ± 23.83 µm and 8.82 ± 0.57 per min in the presence of Mn2+.