Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile that can Catalyze Substrate Cleavage [Enzymology]

January 21st, 2015 by Comeaux, E. Q., Cuya, S. M., Kojima, K., Jafari, N., Wanzeck, K. C., Mobley, J. A., Bjornsti, M.-A., van Waardenburg, R. C. A. M.

Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3′-DNA adducts, such as the 3′phospho-tyrosyl linkage of DNA topoisomerase I (Top1) to DNA. Tdp1 contains two conserved catalytic histidines; a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3′phospho-histidyl intermediate, and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. A Hisnuc to Ala mutant protein is reportedly inactive, while the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of Hisgab to Arg. However, here we report that expression of the yeast HisnucAla (H182A) mutant actually induced Top1-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 Hisgab mutants, including H432N and the SCAN1-related H432R. Moreover, the HisnucAla mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the HisnucPhe mutant was catalytically inactive, and suppressed Hisgab mutant-induced toxicity. These data suggest the activity of another nucleophile when Hisnuc is replaced with residues containing a small side chain (Ala, Asn, Gln), but not with a bulky side chain. Indeed, genetic, biochemical and mass spectrometry analyses show that a highly conserved His, immediately N-terminal to Hisnuc , can act as a nucleophile to catalyze the formation of a covalent Tdp1-DNA intermediate. These findings suggest that the flexibility of Tdp1 active site residues may impair the resolution of mutant Tdp1 covalent phospho-histidyl intermediates, and provides the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.