Mutation of a Conserved Active Site Residue Converts Tyrosyl-DNA Phosphodiesterase I into a DNA Topoisomerase I-dependent Poison

Tyrosyl-DNA Phosphodiesterase I Catalytic Mutants Reveal an Alternative Nucleophile That Can Catalyze Substrate Cleavage

“…yTop1 was constitutively expressed at elevated levels from the GPD promoter YCpGDP-TOP1⅐U (28). The yTop1Y727F catalytic inactive mutant was expressed from the galactose-inducible GAL1 promoter of the YCpGAL1-Top1Y727F⅐U plasmids (7,31). For protein purification from yeast, we first introduced GAL4 coding sequences downstream of the bidirectional 10GAL1 promoter in YEpGAL4 -10GAL1⅐L to enhance galactose-induced expression from the GAL1 promoter.…”

“…Indeed, the catalytic activity of this mutant is similar to that of wild type Tdp1 (23). In contrast, substitution of His gab with residues that contain smaller polar or aliphatic side chains (including Asn, Glu, Leu, Ala, Ser, or Thr) induces an even more severe lethal phenotype, effectively transforming Tdp1 into a potent Top1-dependent cellular toxin, even in the absence of CPT (7,23).…”

“…CPTs, which include the Food and Drug Administration-approved analogs topotecan and irinotecan, reversibly stabilize the covalent Top1-DNA complex to induce S phase-specific lethal DNA lesions (2)(3)(4)(5)(6). More recently, Tdp1 has been shown to process a wide range of other DNA adducts, including 3Ј-modified nucleotides, such as phosphoglycolates and chain-terminating nucleotides, and a 3Ј-phosphohistidyl intermediate formed during Tdp1 catalysis (7)(8)(9)(10)(11)(12)(13).…”

“…Replacing His gab with Arg impairs the resolution of the Tdp1-DNA phosphohistidyl linkage, resulting in a longer-lived covalent Tdp1-DNA reaction intermediate in vitro (10,23). Consequently, SCAN1 lymphoblastoid cells or yeast cells expressing the analogous yeast His gab to Arg (H432R) mutant are more sensitive to CPT-induced DNA lesions or to oxidative DNA damage induced by H 2 O 2 , ␥-irradiation, or bleomycin (7,(23)(24)(25). However, in patients, the SCAN1 phenotype is not evident until the second decade of life, is restricted primarily to the neurodegeneration of cerebellar neurons, and is not associated with an increased risk of cancer or immune deficiency (13).…”

“…For example, mutation of the His nuc to Ala in human and yeast Tdp1 has been reported to inhibit Tdp1 catalytic activity in in vitro assays (12,16,26,27). We therefore hypothesized that introducing the His nuc Ala mutation into the His gab mutant proteins would suppress the enhanced CPT sensitivity of the SCAN1 H432R mutant and the Top1-dependent lethality of the more toxic H432N mutant (7,23,24). Surprisingly, however, we report here that the His nuc Ala substitution (H182A) actually potentiated the cytotoxicity of the His gab mutants and induced a Top1-independent lethal phenotype in combination with the H432N mutant.…”