Abstract 3645: Functional analysis of tyrosyl-DNA phosphodiesterase 1 catalytic residues

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is a highly conserved eukaryotic DNA repair enzyme that hydrolyses 3′phospho-DNA adducts, such as the 3′phospho-tyrosyl intermediate stabilized by camptothecins (CPT) poisoning of DNA topoisomerase I (Top1). Tdp1 catalytic mechanism contains two conserved His residues: The nucleophilic attack of the N-terminal histidine on the Top1-DNA complex releases Top1 and forms a 3′ phosph-histidyl intermediate. The second histidine activates a water molecule to hydrolyze the Tdp1-DNA complex. In humans, a mutation of the second His to Arg (H493R) is associated with the recessive neurodegenerative disease SCAN1. This mutant is defective in processing CPT-induced 3′ phospho-tyrosyl linkages and accumulates Tdp1-DNA complexes in vitro. The analogous yeast SCAN1 mutant (H432R) also enhances cell sensitivity to CPT. However, replacing His432 with Asn (H432N) or Gln (H432Q) induced an even more severe catalytic defect and a Top1-dependent lethality in the absence of CPT. As with SCAN1, these phenotypes were recessive and suppressed by wild-type Tdp1. Our findings suggest that Asn or Gln at this position precludes protonation of the Top1 active site tyrosine phenoxyanion, allowing for the regeneration of the original 3′phospho-tyrosyl linkage. Our recent crystal structures of the H432R and H432N mutants support such a ping-pong reaction mechanism. Surprisingly, however, substituting Ala for the first His (H182A), which should abolish the catalytic activity and therefore the catalytic defects of the Tdp1H432N mutant, did not abolish the Top1-dependent lethality of this Tdp1 mutant. Moreover, expression of the single H182A mutant also induced Top1-dependent toxicity. These data indicate the mutant enzymes are active, leading us to posit that in the absence of His182 but presents of the smaller Ala, the adjacent conserved His181, may rotate into the active site and act as a nucleophile to resolve the Top1-DNA intermediate. Consistent with this model, mutating His181 to Ala suppressed the Top1-dependent lethality of the H182A and H432N mutants. Rotation of His181 is allowed by the small and flexible Ala in the position of His182, thus, a more conservative substitution, such as Phe, should repress His181 rotation and its related toxicity. Indeed, the single His182 to Phe (H182F) mutant showed cell viability. These results indicate that His181 only rotate into the catalytic pocket when position 182 is substituted with a small and flexible residue, such as Ala. Related studies of the corresponding human Top1 and Tdp1 mutants induced similar lethal phenotypes in yeast, supporting a conservation of catalytic mechanism amongst Tdp1 orthologs. Our findings also indicate that the non-conserved N-terminal residues regulate Tdp1-protein interactions, which impact cellular levels of potentially lethal Top1- or Tdp1-DNA covalent complexes, such as those induced by CPT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3645.