The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix - PubMed (original) (raw)

The SRA domain of UHRF1 flips 5-methylcytosine out of the DNA helix

Hideharu Hashimoto et al. Nature. 2008.

Abstract

Maintenance methylation of hemimethylated CpG dinucleotides at DNA replication forks is the key to faithful mitotic inheritance of genomic methylation patterns. UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1) is required for maintenance methylation by interacting with DNA nucleotide methyltransferase 1 (DNMT1), the maintenance methyltransferase, and with hemimethylated CpG, the substrate for DNMT1 (refs 1 and 2). Here we present the crystal structure of the SET and RING-associated (SRA) domain of mouse UHRF1 in complex with DNA containing a hemimethylated CpG site. The DNA is contacted in both the major and minor grooves by two loops that penetrate into the middle of the DNA helix. The 5-methylcytosine has flipped completely out of the DNA helix and is positioned in a binding pocket with planar stacking contacts, Watson-Crick polar hydrogen bonds and van der Waals interactions specific for 5-methylcytosine. Hence, UHRF1 contains a previously unknown DNA-binding module and is the first example of a non-enzymatic, sequence-specific DNA-binding protein domain to use the base flipping mechanism to interact with DNA.

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Figures

Figure 1

Figure 1. Structure of SRA–DNA complex

a, Summary of the SRA–DNA interactions; mc, main-chain-atom-mediated contacts; w, water-mediated hydrogen bonds. Black boxes represent CpG recognition sequence and K495-associated dotted lines represent weak hydrogen bonds. b, The side chains of V451 of the base flipping loop and R496 of the CpG recognition loop are in direct van der Waals contact. c, The two loops—CpG recognition and base flipping—penetrate into the DNA helix from opposite directions. d, The 5mC flips out and binds in a cage-like pocket. e, The surface charge at neutral pH is displayed as blue for positive (20 k_B_T), red for negative (−20 k_B_T), and white for neutral, where _k_B is the Boltzmann's constant and T is the temperature.

Figure 2

Figure 2. Details of SRA–DNA interactions

a, The 5mC•G base pair is shown in the front, and the adjoining G•C base pair is in the back. b, Planar stacking contacts of the extrahelical 5mC with Y471 and Y483 (left image). Omit electron densities, contoured at 4σ and 5σ above the mean, respectively, are shown for omitting 5mC (blue) or the methyl group (red) (right image). c, The hydrogen bond interactions with the polar atoms of 5mC. The double-dotted lines indicate van der Waals contacts with the methyl group of ring carbon C5. d, H450 forms a hydrogen bond from the minor groove side with cytosine of G•C pair at position 5 (see Fig. 1a). e, Network of internal polar interactions centred on residues H447 and S464. Gua, guanine. f, Network of internal charged interactions centred on residues R541 and D560. Distances are shown in angstroms.

Figure 3

Figure 3. Structure of the SRA–DNA specific (_P_212121) and non-specific (_P_6122) complexes

a, The crystal packing interactions in the _P_212121 space group may stabilize the DNA duplex. The extra contacts are made by the helix α4 of a symmetry-related molecule. These interactions are not present in the _P_41212 complex. b, A 12-base-pair duplex containing a single, centrally located unmethylated CG site was used to generate a non-specific complex with the SRA domain. A V451/H450-associated loop approaches the DNA from the minor groove at the junction of two head-to-head DNA molecules.

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