title: Biochemical and structural characterization of the glycosylase domain of MBD4 bound to thymine and 5-hydroxymethyuracil-containing DNA.
abstract: Active DNA demethylation in mammals occurs via hydroxylation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) by the ten-eleven translocation family of proteins (TETs). 5hmC residues in DNA can be further oxidized by TETs to 5-carboxylcytosines and/or deaminated by the Activation Induced Deaminase/Apolipoprotein B mRNA-editing enzyme complex family proteins to 5-hydromethyluracil (5hmU). Excision and replacement of these intermediates is initiated by DNA glycosylases such as thymine-DNA glycosylase (TDG), methyl-binding domain protein 4 (MBD4) and single-strand specific monofunctional uracil-DNA glycosylase 1 in the base excision repair pathway. Here, we report detailed biochemical and structural characterization of human MBD4 which contains mismatch-specific TDG activity. Full-length as well as catalytic domain (residues 426-580) of human MBD4 (MBD4(cat)) can remove 5hmU when opposite to G with good efficiency. Here, we also report six crystal structures of human MBD4(cat): an unliganded form and five binary complexes with duplex DNA containing a T•G, 5hmU•G or AP•G (apurinic/apyrimidinic) mismatch at the target base pair. These structures reveal that MBD4(cat) uses a base flipping mechanism to specifically recognize thymine and 5hmU. The recognition mechanism of flipped-out 5hmU bases in MBD4(cat) active site supports the potential role of MBD4, together with TDG, in maintenance of genome stability and active DNA demethylation in mammals.
authors: Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Gif-sur-Yvette Cedex, F-91198, France. morera@lebs.cnrs-gif.fr
authors: Solange
authors: S
authors: Moréra
authors: Inga
authors: I
authors: Grin
authors: Armelle
authors: A
authors: Vigouroux
authors: Sophie
authors: S
authors: Couvé
authors: Véronique
authors: V
authors: Henriot
authors: Murat
authors: M
authors: Saparbaev
authors: Alexander A
authors: AA
authors: Ishchenko
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D001419:Bacteria / Q000201:enzymology; D020134:Catalytic Domain; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D013941:Thymine / Q000737:chemistry* / Q000378:metabolism; D044502:Thymine DNA Glycosylase / Q000737:chemistry* / Q000378:metabolism
chemical_list: C011865:5-hydroxymethylcytosine; C008055:5-hydroxymethyluracil; D044503:5-Methylcytosine; D010432:Pentoxyl; D003596:Cytosine; D004247:DNA; D004706:Endodeoxyribonucleases; C118712:MBD4 protein, human; D044502:Thymine DNA Glycosylase; D013941:Thymine