Mutation of a Conserved Active Site Residue Converts Tyrosyl-DNA Phosphodiesterase I into a DNA Topoisomerase I-dependent Poison Xiaoping He 1 †, Robert C. A. M. van Waardenburg 2 †, Kerim Babaoglu 1 Allen C. Price 1 , Karin C. Nitiss 2 , John L. Nitiss 2 Mary-Ann Bjornsti 2 ⁎ and Stephen W. White 1 ⁎ 1 Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA 2 Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA Received 25 May 2007; received in revised form 17 July 2007; accepted 19 July 2007 Available online 2 August 2007 Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3′ and 5′ phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1–DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 Å crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H 432 R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1- dependent lethality of Tdp1H 432 N was drug-independent, coinciding with increased covalent Top1–DNA and Tdp1–DNA complex formation in vivo. However, both H 432 mutants were recessive to wild-type Tdp1. Thus, yeast H 432 acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3′ phosphotyrosyl and 3′ phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H 432 N-catalyzed resolution of 3′ phospho-adducts. © 2007 Elsevier Ltd. All rights reserved. Edited by K. Morikawa Keywords: camptothecin; DNA topoisomerase I; enzyme mechanism; Tdp1; X-ray crystallography Introduction The enzyme tyrosyl-DNA phosphodiesterase 1 (Tdp1), initially reported to selectively hydrolyze a phosphotyrosyl linkage formed at the 3′ end of DNA, 1 is encoded by the TDP1 gene and is functionally conserved from yeast to humans. 2,3 In eukaryotes, 3′tyrosyl-DNA adducts arise from the catalytic activity of DNA topoisomerase I (Top1), which unwinds DNA in advance of replication forks and transcription complexes. Top1 transiently cleaves one strand of duplex DNA via the nucleo- philic attack of the active site Tyr on the DNA phosphodiester backbone to yield a 3′phospho- tyrosyl bond. 4–6 The free 5′OH then rotates around the non-scissile strand. The short-lived covalent Top1-DNA intermediate is readily reversed by a second transesterification reaction in which the 5′OH acts as a nucleophile to religate the DNA. Top1 plays a critical role in DNA metabolism, but the covalent Top1-DNA intermediate can be con- verted into DNA lesions that can trigger cell-cycle arrest and cell death. Camptothecin (CPT) targets Top1 by reversibly stabilizing the enzyme–DNA intermediate, and several CPT analogs are effective *Corresponding authors. E-mail addresses: Mary-Ann.Bjornsti@stjude.org; Stephen.White@stjude.org. † X.H. and R.C.A.M.vW. made equal contributions to this work. Present addresses: K. Babaoglu, Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158-2330, USA; A. C. Price, Novartis Institutes for BioMedical Research, Inc., 250 Massachusetts Avenue, Cambridge, MA 02139, USA. Abbreviations used: Tdp1, tyrosyl-DNA phosphodiesterase; Top1, topoisomerase I; CPT, camptothecin; 3′PG, 3′phosphoglycolate; SCAN1, spinocerebellar ataxia with axonal neuropathy; MAD, multiple anomalous dispersion; ssDNA, single-strand DNA. doi:10.1016/j.jmb.2007.07.055 J. Mol. Biol. (2007) 372, 1070–1081 0022-2836/$ - see front matter © 2007 Elsevier Ltd. All rights reserved.