Mutation of a conserved active site residue converts tyrosyl-DNA phosphodiesterase I into a DNA topoisomerase I-dependent poison.
Tyrosyl-DNA phosphodiesterase 1 (Tdp1) catalyzes the resolution of 3' and 5' phospho-DNA adducts. A defective mutant, associated with the recessive neurodegenerative disease SCAN1, accumulates Tdp1-DNA complexes in vitro. To assess the conservation of enzyme architecture, a 2.0 A crystal structure of yeast Tdp1 was determined that is very similar to human Tdp1. Poorly conserved regions of primary structure are peripheral to an essentially identical catalytic core. Enzyme mechanism was also conserved, because the yeast SCAN1 mutant (H(432)R) enhanced cell sensitivity to the DNA topoisomerase I (Top1) poison camptothecin. A more severe Top1-dependent lethality of Tdp1H(432)N was drug-independent, coinciding with increased covalent Top1-DNA and Tdp1-DNA complex formation in vivo. However, both H(432) mutants were recessive to wild-type Tdp1. Thus, yeast H(432) acts in the general acid/base catalytic mechanism of Tdp1 to resolve 3' phosphotyrosyl and 3' phosphoamide linkages. However, the distinct pattern of mutant Tdp1 activity evident in yeast cells, suggests a more severe defect in Tdp1H(432)N-catalyzed resolution of 3' phospho-adducts.
He
Xiaoping
X
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
van Waardenburg
Robert C A M
RCAM
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Babaoglu
Kerim
K
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Price
Allen C
AC
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Nitiss
Karin C
KC
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Nitiss
John L
JL
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Bjornsti
Mary-Ann
MA
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: Mary-Ann.Bjornsti@stjude.org.
White
Stephen W
SW
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA. Electronic address: Stephen.White@stjude.org.
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D018736:DNA Adducts; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D016415:Sequence Alignment; D013379:Substrate Specificity
D018736:DNA Adducts; D029701:Saccharomyces cerevisiae Proteins; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; D004264:DNA Topoisomerases, Type I