Abstract
Tyrosyl-DNA phosphodiesterase I (Tdp1) catalyzes the repair of 3'-DNA adducts, such as the 3'-phosphotyrosyl linkage of DNA topoisomerase I to DNA. Tdp1 contains two conserved catalytic histidines: a nucleophilic His (His(nuc)) that attacks DNA adducts to form a covalent 3'-phosphohistidyl intermediate and a general acid/base His (His(gab)), which resolves the Tdp1-DNA linkage. A His(nuc) to Ala mutant protein is reportedly inactive, whereas the autosomal recessive neurodegenerative disease SCAN1 has been attributed to the enhanced stability of the Tdp1-DNA intermediate induced by mutation of His(gab) to Arg. However, here we report that expression of the yeast His(nuc)Ala (H182A) mutant actually induced topoisomerase I-dependent cytotoxicity and further enhanced the cytotoxicity of Tdp1 His(gab) mutants, including H432N and the SCAN1-related H432R. Moreover, the His(nuc)Ala mutant was catalytically active in vitro, albeit at levels 85-fold less than that observed with wild type Tdp1. In contrast, the His(nuc)Phe mutant was catalytically inactive and suppressed His(gab) mutant-induced toxicity. These data suggest that the activity of another nucleophile when His(nuc) is replaced with residues containing a small side chain (Ala, Asn, and 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 His(nuc), 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 phosphohistidyl intermediates and provide the rationale for developing chemotherapeutics that stabilize the covalent Tdp1-DNA intermediate.
Highlights
Tdp1.Tyrosyl-DNA phosphodiesterase I (Tdp1) repair of adducted DNA involves a covalent enzyme-DNA intermediate, formed and resolved by His263/182 functions as a nucleophile (Hisnuc) and His493/432 acts as a general acid-base (Hisgab) residues
Tdp1 mutants were expressed in a wild type TOP1 strain, with or without subtoxic CPT concentrations, to verify that the lethal phenotypes were a consequence of increased covalent Top1-DNA complexes and not elevated Top1 protein levels per se
In cells deleted for wild type Tdp1, galactose-induced expression of Tdp1H182A suppressed the growth of cells with increased levels of Top1 protein (1Top1) (Fig. 2A and Table 1) and enhanced the CPT sensitivity of cells expressing endogenous levels of Top1
Summary
Tdp repair of adducted DNA involves a covalent enzyme-DNA intermediate, formed and resolved by Hisnuc and Hisgab residues. Tdp contains two conserved catalytic histidines: a nucleophilic His (Hisnuc) that attacks DNA adducts to form a covalent 3-phosphohistidyl intermediate and a general acid/base His (Hisgab), which resolves the Tdp1-DNA linkage. 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 Tdp active site residues may impair the resolution of mutant Tdp covalent phosphohistidyl intermediates and provide the ration-. The more N-terminal catalytic histidine ( referred to as Hisnuc, which is His182 in yeast and His263 human Tdp1) functions in the nucleophilic attack on the 3Ј-phosphoryl-adduct linkage to form a 3Ј-phosphohistidyl bond between Tdp and DNA.
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