title: Structural basis of damage recognition by thymine DNA glycosylase: Key roles for N-terminal residues.
abstract: Thymine DNA Glycosylase (TDG) is a base excision repair enzyme functioning in DNA repair and epigenetic regulation. TDG removes thymine from mutagenic G·T mispairs arising from deamination of 5-methylcytosine (mC), and it processes other deamination-derived lesions including uracil (U). Essential for DNA demethylation, TDG excises 5-formylcytosine and 5-carboxylcytosine, derivatives of mC generated by Tet (ten-eleven translocation) enzymes. Here, we report structural and functional studies of TDG82-308, a new construct containing 29 more N-terminal residues than TDG111-308, the construct used for previous structures of DNA-bound TDG. Crystal structures and NMR experiments demonstrate that most of these N-terminal residues are disordered, for substrate- or product-bound TDG82-308Nevertheless, G·T substrate affinity and glycosylase activity of TDG82-308greatly exceeds that of TDG111-308and is equivalent to full-length TDG. We report the first high-resolution structures of TDG in an enzyme-substrate complex, for G·U bound to TDG82-308(1.54 Å) and TDG111-308(1.71 Å), revealing new enzyme-substrate contacts, direct and water-mediated. We also report a structure of the TDG82-308product complex (1.70 Å). TDG82-308forms unique enzyme-DNA interactions, supporting its value for structure-function studies. The results advance understanding of how TDG recognizes and removes modified bases from DNA, particularly those resulting from deamination.
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors: Christopher T
authors: CT
authors: Coey
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors: Shuja S
authors: SS
authors: Malik
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.
authors: Lakshmi S
authors: LS
authors: Pidugu
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.|University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD 21201, USA.|Center for Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
authors: Kristen M
authors: KM
authors: Varney
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA EPozharskiy@som.umaryland.edu.|University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD 21201, USA.|Center for Biomolecular Therapeutics, Institute for Bioscience and Biotechnology Research, Rockville, MD 20850, USA.
authors: Edwin
authors: E
authors: Pozharski
authors: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA adrohat@som.umaryland.edu.|University of Maryland Marlene and Stewart Greenebaum Cancer Center, Baltimore, MD 21201, USA.
authors: Alexander C
authors: AC
authors: Drohat
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D004789:Enzyme Activation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D044502:Thymine DNA Glycosylase / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D004247:DNA; D044502:Thymine DNA Glycosylase