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Excision of Thymine and 5-Hydroxymethyluracil by the MBD4 DNA

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Excision of Thymine and 5-Hydroxymethyluracil by the MBD4 DNA Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation Hideharu Hashimoto, Emory University Xing Zhang, Emory University Xiaodong Cheng, Emory University Journal Title: Nucleic Acids Research Volume: Volume 40, Number 17 Publisher: Oxford University Press (OUP): Policy C - Option B | 2012-09-01, Pages 8276-8284 Type of Work: Article | Final Publisher PDF Publisher DOI: 10.1093/nar/gks628 Permanent URL: https://pid.emory.edu/ark:/25593/tmsks Final published version: http://dx.doi.org/10.1093/nar/gks628 Copyright information: © 2012 Hashimoto et al. Published by Oxford University Press This is an Open Access work distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/). Accessed September 26, 2021 3:01 PM EDT 8276–8284 Nucleic Acids Research, 2012, Vol. 40, No. 17 Published online 27 June 2012 doi:10.1093/nar/gks628 Excision of thymine and 5-hydroxymethyluracil by the MBD4 DNA glycosylase domain: structural basis and implications for active DNA demethylation Hideharu Hashimoto, Xing Zhang and Xiaodong Cheng* Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, Atlanta, GA 30322, USA Received April 26, 2012; Revised May 24, 2012; Accepted June 4, 2012 ABSTRACT the activation-induced cytidine deaminase (AID) and MBD4 cooperate to demethylate DNA (1). Consistent The mammalian DNA glycosylase—methyl-CpG with a role in DNA demethylation in mammals, AID is binding domain protein 4 (MBD4)—is involved in required to demethylate pluripotency genes during reprogr- active DNA demethylation via the base excision amming of the somatic genome in embryonic stem cell repair pathway. MBD4 contains an N-terminal MBD fusions (6), and AID-deficient animals are less efficient in and a C-terminal DNA glycosylase domain. MBD4 erasure of DNA methylation in primordial germ cells (7). It can excise the mismatched base paired with a is noteworthy that AID promotes 5-methylcytosine (5mC or guanine (G:X), where X is uracil, thymine or 5-hydroxy M) deamination, resulting in thymine (1,8), as well as methyluracil (5hmU). These are, respectively, the de- 5-hydroxymethylcytosine (5hmC or H) deamination, amination products of cytosine, 5-methylcytosine which would produce 5-hydroxymethyluracil (5hmU) (3) (5mC) and 5-hydroxymethylcytosine (5hmC). Here, (Figure 1). There are three mammalian ten eleven transloca- tion (Tet) proteins that convert 5-methylcytosine (5mC or we present three structures of the MBD4 C-terminal M) to 5hmC (9). 5hmC is a constituent of nuclear DNA, glycosylase domain (wild-type and its catalytic mutant present in many tissues and cell types (9–11). The genomic D534N), in complex with DNA containing a G:T or content of 5hmU (<3.5 pmol) is orders of magnitude lower G:5hmU mismatch. MBD4 flips the target nucleotide than that of 5hmC (hundreds of pmols) (10), suggesting that from the double-stranded DNA. The catalytic mutant modification products of 5hmC are probably short lived and D534N captures the intact target nucleotide in the possibly removed by subsequent enzymatic reactions. active site binding pocket. MBD4 specifically recog- The importance of MBD4 for mutation avoidance in nizes the Watson–Crick polar edge of thymine or mammals and in maintaining genome stability is con- 5hmU via the O ,N and O atoms, thus restricting its firmed by an increase in 5mC to T mutations and 2 3 4 À/À activity to thymine/uracil-based modifications while increased occurrence of colon carcinoma in Mbd4 excluding cytosine and its derivatives. The wild-type mice (13). Consistent with this observation, MBD4 is mutated in 26–43% of human colorectal tumors that enzyme cleaves the N-glycosidic bond, leaving the show microsatellite instability (14). Here we show, by ribose ring in the flipped state, while the cleaved 0 means of X-ray crystallography, that the mouse MBD4 base is released. Unexpectedly, the C1 of the sugar glycosylase domain (residues 411–554; Supplementary has yet to be hydrolyzed and appears to form a Figure S1b) binds to a G:T or G:5hmU mismatch in the stable intermediate with one of the side chain context of a CpG dinucleotide. The mismatched nucleo- carboxyl oxygen atoms of D534, via either electrostatic tide (T or 5hmU) is flipped completely out of the DNA or covalent interaction, suggesting a different catalytic helix and is positioned in a binding pocket with Watson– mechanism from those of other DNA glycosylases. Crick polar hydrogen bonds specific for thymine/ uracil-based modifications. INTRODUCTION Mammalian DNA glycosylases have been proposed to be MATERIALS AND METHODS involved in active DNA demethylation via the base excision repair pathway (1–4). MBD4 contains both an N-terminal Expression and purification of MBD4 glycosylase domain methyl-CpG binding domain (MBD) and a C-terminal Hexahistidine-SUMO tagged mouse MBD4 residues DNA glycosylase domain (Supplementary Figure S1a) 411–554 (pXC1064) and its mutants D534N (pXC1088), that acts on G:T and G:U mismatches (5). In zebrafish, K536A (pXC1160) and Y514F (pXC1162) were expressed *To whom correspondence should be addressed. Tel: +1 404 727 8491; Fax: +1 404 727 3746; Email: [email protected] ß The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2012, Vol. 40, No. 17 8277 base pairs (a) (b) mismatches major groove major groove U DNA G C damage G Dnmt T GAPG:AP AID MBD4 G M APOBEC G 5mC BER Tet AID G H APOBEC G 5hm U G:C 5hmC minor groove minor groove Figure 1. A putative pathway of DNA demethylation involving DNA methylation by DNMTs, hydroxylation by Tet proteins, deamination by AID [or members of APOBEC superfamily (12)] and base excision by MBD4 linked to base excision repair (BER). DNA major groove and minor groove sides are indicated. Small arrows in G:M pair and G:T mismatch indicate the hydrogen bond donors and acceptors for 5mC and thymine bases. (a) C, 5mC (M) and its oxidized derivative 5hmC (H) form base pairs with an opposite G, (b) deamination-linked mismatches. in Escherichia coli BL21(DE3)-Gold cells from the concentrated to 0.4 mM at 4C. The complex crystals RIL-Codon plus plasmid (Stratagene). Expression cultures appeared after 1–7 days at 16C under the condition 25% were grown at 37 C until OD600 reached 0.5, shifted to 16 C polyethylene glycol 3350, 200 mM NaCl, 100 mM Bis–Tris– and then induced by adding 0.4 mM isopropyl b-D- HCl pH 5.6. Crystals were cyroprotected by soaking in 1-thiogalactopyranoside. Cell pellets were suspended with mother liquor with 20% ethylene glycol. The X-ray diffrac- 4Â volume of Ni buffer (300 mM NaCl, 20 mM sodium tion data sets for wild-type and D534N mutant co-crystals phosphate pH 7.4, 20 mM imidazole, 1 mM dithiothreitol with DNA were collected at the SER-CAT beamlines and 0.25 mM phenylmethylsulfonyl fluoride) and sonicated 22BM-E and 22ID-D, respectively, and processed using for 5 min (1 s on and 2 s off). The lysate was clarified by HKL2000 (15). The structures were solved by molecular centrifugation twice at 38 000g for 30 min. His6-SUMO replacement using PHENIX (16), using the mouse MBD4 fusion proteins were isolated on a nickel-charged chelating glycosylase domain apo structure (PDB 1NGN) (17) as the column (GE Healthcare). The His6–SUMO tag was cleaved searching model. Electron density for DNA was easily by ULP-1 protease (16 h at 4C), leaving two extraneous interpretable, using the model-building program Coot (18). N-terminal amino acids (HisMet). The cleaved protein was PHENIX/Refinement scripts were used for refinement, and loaded onto tandem ion exchange HiTrap-Q and HiTrap-SP the statistics shown in Supplementary Table S1 were columns (GE-Healthcare), eluted from the SP column and calculated for the entire resolution range. The Rfree and further purified by Superdex 75 (16/60) in the presence of Rwork values were calculated for 5% (randomly selected) 500 mM NaCl. and 95%, respectively, of observed reflections. The struc- tures were solved, built and refined independently. Crystallography of MBD4 and DNA complexes DNA glycosylase activity assay For co-crystallization, the MBD4 proteins (WT or D534N mutant) and annealed G:X mismatch oligonucleotide (50-TC MBD4 glycosylase activity assays were performed using AGCGCATGG-30 and 50-CCATGXGCTGA-30;where various oligonucleotides labeled with 6-carboxy-fluor X = T or 5hmU, synthesized by Sigma or New England escein (FAM), and the excision of the target base was Biolabs, respectively) were mixed at a 1:1 ratio and diluted monitored by denaturing gel electrophoresis following 5Â with buffer (20 mM HEPES pH 7.0, 1 mM dithiothreitol) NaOH hydrolysis (Supplementary Figure S1c). In to reduce the salt concentration to 100 mM, and then Figure 2a, MBD4 protein (0.5 mM) and equal amount of 8278 Nucleic Acids Research, 2012, Vol. 40, No. 17 double-stranded FAM labeled 32-bp duplexes were mixed in For enzymatic reactions under single turnover conditions 20 ml nicking buffer (10 mM Tris–HCl, pH 8.0, 1 mM (Figure 2b), the FAM-labeled 32-bp duplexes (0.25 mM) and EDTA, 0.1% BSA) and incubated at 37C for 30 min. 10-fold excess of MBD4 catalytic domain (2.5 mM) were Reactions were stopped by adding 2 mlof1NNaOH, incubated for 0–10 min at room temperature (22C) and boiled for 10 min, and 20 ml of loading buffer (98% processed as described earlier.
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