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 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+.
Cheng
Ting-Jen
TJ
Keck Graduate Institute of Applied Life Sciences, Claremont, CA 91771, USA.
Rey
Peter G
PG
Poon
Thomas
T
Kan
Chen-Chen
CC
D000595:Amino Acid Sequence; D003124:Colorimetry / Q000379:methods; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013938:Thymidine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D015003:Yeasts / Q000201:enzymology
D011994:Recombinant Proteins; C032579:thymidine 5'-4-nitrophenyl phosphate; D013938:Thymidine Monophosphate; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C122147:tyrosyl-DNA phosphodiesterase