title: Biochemical characterization of human tyrosyl-DNA phosphodiesterase 2 (TDP2/TTRAP): a Mg(2+)/Mn(2+)-dependent phosphodiesterase specific for the repair of topoisomerase cleavage complexes.
abstract: TDP2 is a multifunctional enzyme previously known for its role in signal transduction as TRAF and TNF receptor-associated protein (TTRAP) and ETS1-associated protein 2 (EAPII). The gene has recently been renamed TDP2 because it plays a critical role for the repair of topoisomerase II cleavage complexes (Top2cc) and encodes an enzyme that hydrolyzes 5'-tyrosine-DNA adducts that mimic abortive Top2cc. Here we further elucidate the DNA-processing activities of human recombinant TDP2 and its biochemical characteristics. The preferred substrate for TDP2 is single-stranded DNA or duplex DNA with a four-base pair overhang, which is consistent with the known structure of Top2cc or Top3cc. The k(cat)/K(m) of TDP1 and TDP2 was determined. It was found to be 4 × 10(5) s(-1)M(-1) for TDP2 using single-stranded 5'-tyrosyl-DNA. The processing of substrates as short as five nucleotides long suggests that TDP2 can directly bind DNA ends. 5'-Phosphodiesterase activity requires a phosphotyrosyl linkage and tolerates an extended group attached to the tyrosine. TDP2 requires Mg(2+) or Mn(2+) for efficient catalysis but is weakly active with Ca(2+) or Zn(2+). Titration with Ca(2+) demonstrates a two-metal binding site in TDP2. Sequence alignment suggests that TDP2 contains four conserved catalytic motifs shared by Mg(2+)-dependent endonucleases, such as APE1. Substitutions at each of the four catalytic motifs identified key residues Asn-120, Glu-152, Asp-262, and His-351, whose mutation to alanine significantly reduced or completely abolished enzymatic activity. Our study characterizes the substrate specificity and kinetic parameters of TDP2. In addition, a two-metal catalytic mechanism is proposed.
authors: Laboratory of Molecular Pharmacology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, MD 20892, USA.
authors: Rui
authors: R
authors: Gao
authors: Shar-yin N
authors: SY
authors: Huang
authors: Christophe
authors: C
authors: Marchand
authors: Yves
authors: Y
authors: Pommier
mesh_terms: D020816:Amino Acid Motifs; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002413:Cations, Divalent; D018736:DNA Adducts / Q000737:chemistry* / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D008345:Manganese / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases; D013379:Substrate Specificity / Q000502:physiology; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism
chemical_list: D000951:Antigens, Neoplasm; D002413:Cations, Divalent; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D009097:Multienzyme Complexes; D009687:Nuclear Proteins; D014157:Transcription Factors; D008345:Manganese; D010727:Phosphoric Diester Hydrolases; C474866:TDP2 protein, human; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D008274:Magnesium