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 (H432R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H432N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H432 mutants were recessive to wild-type Tdp1. Thus, yeast H432 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 Tdp1H432N-catalyzed resolution of 3′ phospho-adducts. © 2007 Elsevier Ltd. All rights reserved.