title: Tyrosyl-DNA phosphodiesterase I catalytic mutants reveal an alternative nucleophile that can catalyze substrate cleavage.
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.
authors.affiliation: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Evan Q
authors.initials: EQ
authors.lastname: Comeaux
authors.affiliation: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Selma M
authors.initials: SM
authors.lastname: Cuya
authors.affiliation: the University of Alabama at Birmingham Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Kyoko
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, and.
authors.forename: Nauzanene
authors.initials: N
authors.lastname: Jafari
authors.affiliation: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Keith C
authors.initials: KC
authors.lastname: Wanzeck
authors.affiliation: the Department of Surgery, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: James A
authors.initials: JA
authors.lastname: Mobley
authors.affiliation: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294.
authors.forename: Mary-Ann
authors.initials: MA
authors.lastname: Bjornsti
authors.affiliation: From the Department of Pharmacology and Toxicology, University of Alabama at Birmingham, Birmingham, Alabama 35294, rvanwaar@uab.edu.
authors.forename: Robert C A M
authors.initials: RC
authors.lastname: van Waardenburg
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D018736:DNA Adducts / Q000737:chemistry* / Q000235:genetics; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics*; D020754:Spinocerebellar Ataxias / Q000235:genetics / Q000473:pathology
chemical_list: D018736:DNA Adducts; D046912:Multiprotein Complexes; D050505:Mutant Proteins; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; D004264:DNA Topoisomerases, Type I; C528909:TOP1 protein, human
keywords: DNA Damage; DNA Repair; DNA Topoisomerase; Enzyme Mechanism; Enzyme Mutation; Protein-DNA Covalent Complexes; Spinocerebellar Ataxia with Axonal Neuropathy 1; Tyrosyl-DNA Phosphodiesterase I