tailieunhanh - Báo cáo Y học: 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, ., Yao, ., Robertson, . & Nash, . (1999) Science 286, 552–555]. | Eur. J. Biochem. 269 3697-3704 2002 FEBS 2002 doi Kinetic studies of human tyrosyl-DNA phosphodiesterase an enzyme in the topoisomerase I DNA repair pathway Ting-Jen Cheng1 Peter G. Rey2 Thomas Poon2 and Chen-Chen Kan1 1Keck Graduate Institute of Applied Life Sciences CA USA 2W. M. Keck Science Center Claremont McKenna Pitzer and Scripps Colleges CA USA 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 . Yao . Robertson . Nash . 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 . Cham-poux . 2001 Proc. Natl Acad. Sci. USA 98 1200912014 and Davies . Interthal H. Champoux . Hol . 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 -phos-phate as substrate. This assay system is able to detect enzymatic activity at enzyme concentrations as low as 15 nM. Purified recombinant human TDPNA39 cleaved p-nitro-phenyl-thymidine-3 -phosphate with Km and kcat values of M and per min in the presence of Mn2 . Keywords tyrosyl-DNA phosphodiesterase topoisomerase I phospholipase D high-throughput screening. In eukaryotic cells DNA topoisomerase I Topo I is an enzyme that relaxes DNA supercoiling and .