Structural insight into maintenance methylationby mouse DNA methyltransferase 1 (Dnmt1)Kohei Takeshitaa,1, Isao Suetakeb,1, Eiki Yamashitaa, Michihiro Sugaa,2, Hirotaka Naritaa,Atsushi Nakagawab,3, and Shoji Tajimab,3

aLaboratory of Supramolecular Crystallography, and bLaboratory of Epigenetics, Institute for Protein Research, Osaka University, 3-2 Yamadaoka,Suita, Osaka 565-0871, Japan

Edited* by Peter Walter, University of California, San Francisco School of Medicine, San Francisco, CA, and approved March 24, 2011 (received for reviewDecember 29, 2010)

Methylation of cytosine in DNA plays a crucial role in developmentthrough inheritable gene silencing. The DNA methyltransferaseDnmt1 is responsible for the propagation of methylation patternsto the next generation via its preferential methylation of hemi-methylated CpG sites in the genome; however, how Dnmt1 main-tains methylation patterns is not fully understood. Here we reportthe crystal structure of the large fragment (291–1620) of mouseDnmt1 and its complexes with cofactor S-adenosyl-L-methionineand its product S-adenosyl-L-homocystein. Notably, in the absenceof DNA, the N-terminal domain responsible for targeting Dnmt1 toreplication foci is inserted into the DNA-binding pocket, indicatingthat this domain must be removed for methylation to occur. Uponbinding of S-adenosyl-L-methionine, the catalytic cysteine residueundergoes a conformation transition to a catalytically competentposition. For the recognition of hemimethylated DNA, Dnmt1 isexpected to utilize a target recognition domain that overhangs theputative DNA-binding pocket. Taking into considerations the re-cent report of a shorter fragment structure of Dnmt1 that the CXXCmotif positions itself in the catalytic pocket and prevents aberrantde novo methylation, we propose that maintenance methylation isa multistep process accompanied by structural changes.

maintenance DNA methylation ∣ X-ray crystallography ∣multidomain structure

In mammals, genomic DNA is often methylated at the fifthposition of the cytosine base in CpG sequences (1). This DNA

methylation, which is one of the major epigenetic modifications,plays a crucial role in development, genome stability, X-chromo-some inactivation, and silencing of retrotransposons (2–4). DNAmethyltransferases (Dnmts) catalyze the transfer of a methylgroup to the fifth position of cytosine bases in DNA. Mammaliangenomes carry three distinct active Dnmt genes. Two of the threegenes, Dnm3a and Dnmt3b, encode enzymes that show activitytoward unmethylated DNA (5, 6) and are responsible for creatingglobal DNA methylation patterns during embryogenesis andgametogenesis (7, 8). Once the DNA methylation patterns areestablished, they are maintained by Dnmt1 encoded by theDnmt1 gene, which ensures the transmission of lineage-specificDNA methylation patterns during replication (9). Dnmt1 prefer-entially methylates the hemimethylated state of DNA thatappears just after replication or repair. To do this, Dnmt1 inter-acts both with proliferating cell nuclear antigen (PCNA), a factorthat is a prerequisite for replication (10), and Np95/Uhrf1, a fac-tor that is necessary for the maintenance of DNA methylationand that binds hemimethylated DNA at replication foci (11–14).Knockout ofDnmt1 in mice leads to embryonic lethality at a stagecorresponding to global methylation in the genome (9).

All of the DNA methyltransferases identified to date utilizeS-adenosyl-L-methionine (AdoMet) as the methyl group donor.The reaction mechanism of cytosine-C5 methylation has beenanalyzed for the prokaryotic DNA (cytosine-C5)-methyltransfer-ase M.HhaI (15). A key catalytic process is the nucleophilic attackof the enzyme on the sixth carbon of the target cytosine. This

attack is made by the thiol group of the cysteine residue in theconserved PCQ motif (motif IV) of the methyltransferase (Fig. S1).The reaction is catalyzed by protonation of the N3 position ofthe cytosine by the glutamate in the ENV motif (motif VI).

Dnmt1 is a large single polypeptide, and its primary sequence ishighly conserved among various species. MouseDnmt1 comprises1620 amino acid residues with a size of 180 kD. The C-terminalcatalytic domain (residues 1125–1620) and the remaining N-term-inal regulatory domain (residues 1–1111) are connected by a flex-ible KG-repeat (residues 1112–1124) (Fig. 1A). In the N-terminalregion, the N-terminal 243 residues form an independent domainthat serves as a platform (16) for the binding of many proteins orDNA including PCNA (10, 16–20).

In the present study, we crystallized a large fragment of Dnmt1lacking only the N-terminal 1–290 residues (Dnmt1(291–1620))(Fig. 1B) that selectively methylates hemimethylated DNA invitro (21). Recently, the crystal structure of a Dnmt1 fragmentencompassing amino acids 550–1602 in complex with unmethy-lated DNA has been reported (22). Based on both structures,we propose that multiple changes occur during maintenancemethylation.

Results and DiscussionRFTS Is Inserted into the Putative DNA-Binding Pocket of Dnmt1. Wehave succeeded in crystallizing a Dnmt1 fragment comprising theamino acid sequence 291–1620 and determined the followingthree crystal structures: the substrate unbound free form, and theS-adenosyl-L-homocystein-(AdoH)bound and AdoMet-boundforms (Table S1). The structures were successfully modeled aselectron density maps (Fig. S2A), and the amino acid assignmentwas confirmed by methionine sites (Fig. S2B). The whole crystalstructure of Dnmt1(291–1620) showed a distinct multidomainstructure comprising the replication foci targeting sequence(RFTS), a zinc-finger-like (CXXC) motif, two tandemly con-nected bromo-associated homology (BAH) domains, and thecatalytic domain (Fig. 1B). The multiple domains in the N-term-inal region surround and make contact with the C-terminalcatalytic domain.

Author contributions: A.N. and S.T. designed research; K.T., I.S., M.S., H.N., and S.T.performed research; K.T., I.S., E.Y., A.N., and S.T. analyzed data; and K.T., I.S., A.N., andS.T. wrote the paper.

The authors declare no conflict of interest.

*This Direct Submission article had a prearranged editor.

Data deposition: Atomic coordinates and structure factors for the reported crystalstructure have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes3av4, 3av5, and 3av6).

See Commentary on page 8919.1K.T. and I.S. contributed equally to this work.2Present address: Vollum Institute, Oregon Health and Science University, 3181 SW SamJackson Park Road, Portland, OR 97239.

3To whom correspondence may be addressed. E-mail: tajima@protein.osaka-u.ac.jp oratsushi@protein.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019629108/-/DCSupplemental.

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Among the N-terminal domains, the position of RFTS isunique. It is apparently inserted deeply into the DNA-bindingpocket, where it forms several hydrogen bonds with the catalyticdomain (Fig. 2A). The electrostatic potential of the RFTS andthe catalytic domains at the interface is a negative and positivenet charge, respectively (Fig. S3). Because the RFTS domain in-teracts via limited numbers of contacts with the next molecules inthe crystal structure, it is unlikely that the crystallization affectedthe orientation of the RFTS domain. It has been reported thatthe RFTS domain is required for recruitment of Dnmt1 to repli-cation foci (23). Interestingly, however, based on our presentstructure, DNA cannot bind to the catalytic pocket owing to sterichindrance from RFTS, which sits in the pocket.

It is puzzling that, with the RFTS domain inserted deeply intothe DNA-binding pocket where hemimethylated DNA is ex-pected to fit, Dnmt1(291–1620) still shows methylation activitytoward hemimethylated DNA (21). It is reasonable to speculatethat unless the inserted RFTS domain is released, DNA cannotaccess the catalytic center. The linker connecting the RFTSdomain and CXXC motif is interacting with the PCQ loop at

the catalytic center by the hydrophobic interaction of the linkeramino acid residues (F631, F634, and F635) with the ones (Y1243and F1246) following the PCQ loop (Fig. 2B and Fig. S4). Theinteraction between the linker and the PCQ loop contributesto narrowing the entrance of the DNA-binding pocket and toanchoring the RFTS domain to the DNA-binding pocket, andconsequently the catalytic center is completely masked.

It is reasonable to expect that significant activation energy isnecessary to change the positions of the RFTS domain and theCXXC motif to allow DNA binding and subsequent DNAmethy-lation activity. Recombinant Dnmt1(291–1620) demonstrateshigher DNA methylation activity toward hemimethylated DNAthan unmethylated ones (21). In addition, Dnmt1(602–1620), inwhich the RFTS coding sequence is deleted, also showed a spe-cificity toward hemimethylated DNA similar to that of Dnmt1(291–1620) containing RFTS (Fig. S5). Intriguingly, the activa-tion energy of Dnmt1(291–1620) for the DNA methylation reac-tion was about threefold larger than that of Dnmt1(602–1621)(Fig. 2C).

Recently, the crystal structure of a Dnmt1 fragment compris-ing residues 650–1602 in complex with unmethylated DNA hasbeen reported (22). The reported Dnmt1 fragment lacks RFTSdomain. In our structure, different from the structure by Songet al. (22), the CXXC motif sits where unmethylated DNA binds.One of the two zinc ions and the interacting peptide chain in theCXXC motif are not seen in our present structure. Superimposi-tion of our structure and that by Song et al. is shown in Fig. 3. Forthe binding of substrate DNA, the RFTS domain must change itsposition. In addition, the CXXC motif is pushed toward the cat-alytic center in the complex with unmethylated DNA, in whichposition the CXXC motif cannot bind the hemimethylated formof CpG (22). The sequence following the CXXC motif formsan α-helix in our structure but forms a loop in Song’s (22).The interaction of the CXXC motif with DNA may affect the lin-ker between RFTS and CXXC and possibly induces a conforma-tional change in Dnmt1. Because almost no significant structuraldifference in the catalytic domain including PCQ loop is foundbetween our structure and that by Song’s (see Fig. 3), the bindingof unmethylated DNA may thereby affect exclusively the releaseof the RFTS domain from the DNA-binding pocket. For DNAmethylation, displacement of RFTS from the catalytic centerand movement of the CXXC motif must require energy, whichsuggests that there is a mechanism to regulate the insertion ofRFTS into the catalytic domain. The interaction with the targetDNA via the CXXC motif with its net positive charge (Fig. S6)can be a crucial step for the initiation of the release of the RFTSdomain from the DNA-binding pocket for the DNA methylationactivity.

AdoMet Induces a Position Change of the Cysteine Residue in the PCQLoop at the Catalytic Center. The 10 motifs characteristic to DNA

1 291 1620

Linker of helices loop KG repeat

N-terminalplatform

RFTS CXXC BAH1 BAH2Catalytic domian650

TRD

RFTS

CXXC

BAH1 BAH2

180°

A

B

I IV IV VIII IX X

Fig. 1. Multidomain structure of mouse Dnmt1(291–1620). (A) Replicationfoci targeting sequence (RFTS), zincfinger-like motif (CXXC), bromo-asso-ciated homology domain 1 (BAH1) and 2 (BAH2), and catalytic domain areschematically illustrated. The catalytic domain comprising 10 conserved mo-tifs (I ∼ X), and target recognition domain (TRD) between the motifs VIII andIX. The numbers of the amino acid residues are indicated. (B) Ribbonmodel ofmouse Dnmt1(291–1620). Around the C-terminal catalytic domain (blue), theother domains including the RFTS (magenta), CXXC motif (cyan), and twoBAH domains BAH1 (green) and BAH2 (orange) are shown. Four zinc ionsare shown in red spheres. All of the zinc ions are in amotif similar to Zn-fingermotif (Fig. S10). The KG-repeat (1112–1124) linker connecting the N-terminalregion and the C-terminal catalytic domain is in a flexible structure as thedensity map showed disorder.

D532

R1576

K1537

E531

S1495

H1504

T1505

D554

A594

L593

RFTS

Catalyticdomain

Linker

CXXC

Catalytic domain

PCQ

RFTS

0.1

1

10

3.25 3.35 3.451/T (10 )-3

DN

A m

ethy

latio

n ac

tivity

A B C

Fig. 2. The RFTS domain plugs into the DNA-binding pocket. (A) The RFTS domain is positioned in the DNA-binding pocket of Dnmt1 and stabilized by severalhydrogen bonds in the catalytic domain. (B) The linker (brown) between the RFTS domain (deep red) and the CXXCmotif (light blue) interacts with the catalyticdomain (dark blue) by hydrophobic forces to maintain the position of the PCQ loop (see Fig. S7). C. Fitting to Arrhenius equation of the DNA methylationactivity of Dnmt1(291–1620) (cyan diamonds) containing the RFTS domain and Dnmt1(602–1620) (magenta squares) lacking the RFTS domain. The activationenergies of Dnmt1(291–1620) and Dnmt1(602–1620) were calculated to be 110 and 30 kJ∕mol, respectively.

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(cytosine-C5)-methyltransferases (24) are conserved in the cata-lytic domain of Dnmt1 (Fig. S1). The three-dimensional structureof Dnmt1 shows that the spatial arrangement of the motifs is si-milar to the reported structure of M.HhaI (PDB ID code 5mht)(Fig. S7A). It is reasonable to assume that the DNA methylationmechanism of Dnmt1 is similar to that of M.HhaI (15), and thusthe C1229 in the PCQ loop is expected to form a covalent bondwith the sixth carbon of the target cytosine base. In M.HhaI, theside chain conformation of C81 in the PCQ loop at the catalyticcenter is facing away from the double-stranded DNA, in the pre-sence of AdoMet (25). The C81 turns to approach the cytosinebase to be methylated only when it binds to DNA (26). From this,O’Gara et al. proposed an ordered mechanism in which DNAbinding precedes the binding of AdoMet (27). Similar to M.HhaI,the side chain of C1229 in the PCQ loop of Dnmt1(291–1620)faces away from the putative DNA-binding site in the absenceand presence of AdoH (Fig. 4A and Fig. S7B). Notably, the sidechain of C1229 in the PCQ loop turns to face the putative DNA-binding pocket, which is the catalytically competent position cor-responding to the side chain of C81 of M.HhaI, when it binds toDNA (Fig. 4B). The phenyl group of F1232 is apparently pushingthe side chain of C1229 into its position (Fig. S8). Distinct fromM.HhaI, the present study clearly shows that prebinding of Ado-Met to Dnmt1 shifts the position of the side chain of C1229 inthe PCQ loop close to the expected position of the flipped-outcytosine. Thus, we propose that binding of AdoMet shifts theDnmt1 structure to a state that is ready to receive hemimethy-lated DNA from Np95/Uhrf1, a factor that specifically bindsto hemimethylated DNA for the maintenance methylation in vivo(11). As soon as the methyl group is transferred from AdoMetto the cytosine base to produce AdoH, the side chain of C1229

immediately faces away. The binding of AdoMet does not changethe overall structure of Dnmt1 including the catalytic domainother than the cysteine residue in the PCQ loop. A possible rea-son for the AdoMet-induced change in the position of the sidechain of C1229 might be that the DNA-binding pocket of Dnmt1is slightly narrower than that of M.HhaI when it is not bindingDNA (Fig. S7B). As discussed above, the amino acid residuesfollowing the PCQ loop are interacting with the linker betweenthe RFTS domain and the CXXC motif (Fig. S4). These forcesthat pull the PCQ loop toward the DNA-binding pocket mightaffect the position change of C1229 induced by AdoMet bindingin Dnmt1.

Recognition of Hemimethylated DNA. The substrate specificity ofDnmt1, which preferentially methylates hemimethylated DNA,is unique among DNA (cytosine-C5)-methyltransferases (28). Inbacterial DNA (cytosine-C5)-methyltransferases, the target re-cognition domain (TRD), which recognizes the sequence to bemethylated, is located between motifs VIII and IX (29–31)(Fig. S1). Intriguingly, the TRD of Dnmt1 is unusually largeas compared with those of bacterial enzymes (24). This largeTRD overhangs the putative DNA-binding pocket where dou-ble-stranded DNA fits for the methylation reaction (Fig. 5A).Because the 10 motifs comprising the catalytic center accessthe target cytosine to be methylated from one side, it seems rea-sonable that the TRD that overhangs the DNA-binding pocket

90°RFTS

PCQ-loop

PC

Q

RFTS

Fig. 3. Model of conformational changes that are predicted to be induced by DNA binding. Superimposition of the free-form structure including the RFTSdomain (blue, PBD ID code 3av4) and the complex with unmethylated DNA structure (yellow, PDB ID code 3pt6). The CXXC motifs (dark blue) and the linkerconnecting the RFTS domain and CXXCmotif (light blue) are indicated in deep colors, and the rest sequences are in pale colors. The RFTS domain (pale gray) andunmethylated DNA (pale yellow) are shown in surface model. The sequences involved in the interaction of the linker connecting the RFTS domain and CXXCmotif with the PCQ loop at the catalytic center are magnified.

C1229

P1228

Q1230

CH3

C5C6

C1229

AdoMet AdoH

A B

Fig. 4. AdoMet turns the side chain of the cysteine in the PCQ loop towardthe target cytosine. (A) Superimposition of the PCQ loop of Dnmt1 in its freeform (green), with AdoH (orange), and with AdoMet (blue). (B) Binding ofAdoMet to the catalytic site induces the side chain of C1229 to turn towardthe target cytosine (Left). C1229 is expected to bind to C6 and the methyl-group of AdoMet binds to C5, of the cytosine (red arrows). After transfer ofthe methyl-group to the fifth position of cytosine, C1229 turns back to theinactive AdoH-binding form (Right). The position of cytosine was taken fromPDB ID code 5mht, the structure of M.HhaI complex with methylated DNAand AdoH.

Fig. 5. The TRD of Dnmt1 overhangs the putative DNA-binding pocket. (A)Superimposition of the catalytic domain of Dnmt1withM.HhaI. The positionsof the TRD of Dnmt1 (light blue) and the hemimethylated double-strandedDNA fitted form of the 10 motifs (blue) of the catalytic domain of Dnmt1(291–1620) superimposed with M.HhaI (PDB ID code 5mht) (pink) are shown.(B) DNA-fitted model of Dnmt1(291–1620) showing thatW1512 is close to themethylated cytosine base of the hemimethylated DNA when the methylatedcytosine (5MC) stays inside the double-stranded DNA, whereasW1500 is closeto when the 5MC is flipped out.

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from the other side is responsible for recognizing the methylatedcytosine base in the hemimethylated CpG sequence. By mimick-ing the three-dimensional structure of the M.HhaI complexwith DNA, which is the active form in complex with DNA andin which the target cytosine base is flipped outside the double-stranded DNA, we fitted DNA into the catalytic pocket ofDnmt1. According to the resultant model, W1512 is close tothe methylated cytosine that is opposite the target cytosine of thehemimethylated CpG (Fig. 5B). In addition, W1500 becomesclose to this methylated cytosine when the base is flipped outsidethe double-stranded DNA (Fig. 5B). In accordance with thisobservation, we replaced W1500 and W1512 with A or L anddetermined the DNA methylation activity of the mutants. All themutants of Dnmt1 showed almost complete abolition of DNAmethylation activity toward both hemimethylated and unmethy-lated DNA (Fig. S9). Because we expected that the position ofthe methylated CpG would be fixed, either facing the antisenseCpG or in a flipped-out configuration, it was surprising that themutations at W1500 and W1512 both inhibited the DNA methy-lation activity. This observation may suggest that both residuesare involved in recognition of the target hemimethylated DNAfor methylation activity.

Maintenance Methylation Is Accompanied by Structural Changes. Thestructure reported by Song et al. (22) showed the Dnmt1(550–1602) complex with DNA, which is positioned away from the cat-alytic center, leading them to propose that the deduced structurerepresents the auto-inhibition form of Dnmt1 protecting from denovo DNAmethylation. In contrast, our present crystal structure,although not containing DNA, but including the RFTS and themethyl group donor AdoMet, shows three striking features. First,the RFTS is inserted into the catalytic pocket such that DNAcannot access the catalytic center. Second, the CXXC motif is ina position where the DNA binds in the structure reported by Songet al. (22). Last, the complex with AdoMet causes C1229 to fliptoward the target cytosine. This residue is expected to form acovalent bond with the sixth position of the target cytosine base.The results clearly indicate that multiple structural changes musttake place for faithful maintenance DNA methylation.

Materials and MethodsExpression and Purification of Dnmt1(291–1620) and Dnmt1(602–1620). MouseDnmt1 was cloned into baculovirus in accordance with the manufacturer’sinstructions and was used to infect to Sf9 cells. The expressed Dnmt1 proteinwas purified as described elsewhere (21) and then further purified by size-exclusion chromatography in 0.35 M NaCl, 20 mMHEPES-Na (pH 7.0), concen-trated to 9 mg∕mL, and then used for crystallization. To prepare seleno-methionine (Se-Met) labeled Dnmt1, Sf9 cells were collected 18 h after infec-tion, washed with sterilized PBS, and then transferred into Sf900II mediumdepleted of methionine and cysteine (Invitrogen), and supplemented with100 mg∕L Se-Met and 150 mg∕L L-cysteine. The cells were collected after48 h, and Se-Met-labeled Dnmt1 was purified by the identical protocol usedfor native protein.

Crystallization Crystals were obtained by vapor diffusion by mixing equalvolumes of protein solution (6.5 mg∕mL) and reservoir solution containing0.35 M NaCl, 2% Tascimate (Hampton Research) 20 mM TCEP-HCl (HamptonResearch), 18–20% PEG3350, and 100 mM Tris-HCl (pH 9.0). The incubationtemperature was changed stepwise from 28 to 23 °C. In brief, the solutionwas initially incubated at 28 °C for 6 to 12 h, and then transferred to 23 °Cfor crystal growth. The crystals that formed were transferred to a solution of

PEG3350 2% higher than the reservoir solution to stabilize the crystal. Crys-tals were treated by the gradual addition of PEG 200 to a final concentrationof 20% and flash frozen in liquid nitrogen. To determine the initial phase ofdiffraction, the crystals were soaked with 5 mM Ta6Br14 derivative for 2 h.

X-ray Data Collection Data sets were collected on BL44XU at SPring-8 with aDIP6040 imaging-plate detector (Bruker AXS) and a MX225-HE chargecoupled device detector (Raynonix). The native data was collected at 2.75-Å resolution. Isomorphous derivative datasets of tantalum cluster derivativecrystal was acquired by tuning X-rays at 1.2454 Å (Derivative I: Ta6Br14).Diffraction data of Se-Met derivative crystals were collected with X-rays of0.90000 Å (remote), 0.97894 Å (peak), and 0.97954 Å (edge) (Derivative II:Se-Met). Anomalous difference Fourier maps of all datasets (Native andDerivative I, II) showed clear peaks at the same positions and suggestedthe existence of anomalous scatterers that specifically bound to the mole-cule. These peaks were assumed to be zinc ions, because it is predicted thatzinc binding motifs (CXXC motif) exist in the primary structure of Dnmt1.Next three datasets of native crystals were collected at 0.9000 Å (remote),1.28220 Å (peak), and 1.28309 Å (edge) for phase determination by theanomalous effect of zinc ions (Derivative III: Zinc dataset). The complex crystalwith AdoMet or SAH was prepared by soaking with crystallization buffer andcryoprotectant including cofactor ligand, of which the final concentrationwas 100 μM. Two complex datasets for AdoMet (Native AdoMet) and SAH(Native SAH) were collected at 3.10 Å and 3.25 Å resolution, respectively.All X-ray experiments were performed at 90 K. These SPring-8 diffractiondata were processed and scaled with HKL2000 (32). The native crystalsand derivative crystals belonged to the space group P21212. The experimen-tal conditions and statistics of intensity data acquisition are given in Table S1.

Structure Determination For the initial phase determination, the heavy atomsites of the Ta6Br14 clusters were determined by using the difference Patter-son map calculated with Native data and Derivative I data at 6-Å resolution,and the SIRASmethodwas applied. The anomalous effects of selenium atomsin the Se-Met derivative and the intrinsic five zinc ions were used for phasedetermination by the MAD method with SHARP (33). Twenty-three out of 28methionine sites in Dnmt1(291–1620) were identified by selenium peakshigher than 3.5σ in the electron density distribution in the anomalous differ-ence Fourier map. Five zinc sites were identified from the anomalous differ-ence Fourier map. Model building was performed by using the program Coot(34), and structural refinement was carried out with the programs REFMAC(35) and BUSTER (36). Crystallographic R and Rfree for 5% of the reflectionsexcluded from the refinement were calculated to monitor the structuralrefinement procedures. The results of the structural analysis are summarizedin Table S1. The free form structure of the final R and Rfree values were 23.5%and 26.9%, respectively. The main-chain dihedral angles for 90.6% were inthe favored and 1.6% were in outlier of the Ramachandran plot as definedin MolProbity (37). These refinement statistics of the AdoH and AdoMetcomplexes are given in Table S1. The refined structure was validated by usingthe programMolProbity (37). All molecular graphics were created with Pymol(DeLano Scientific, http://www.pymol.org).

Determination of DNA Methylation Activity The DNA methylation activity wasdetermined as described elsewhere (20). For determination of the activationenergy, Dnmt1(291–1620) and Dnmt1(602–1620) were incubated at 20, 30,and 37 °C for 1 h.

ACKNOWLEDGMENTS.We wish to thank Ms. Yumiko Yamagami for construct-ing the baculoviruses coding Dnmt1 and expression in sf9 cells, and Ms. KeikoShinohara for purification of Dnmt1. This work was partly supported byGrants-in-Aid for Scientific Research B by the Japan Society for the Promotionof Science (S.T.), by the Japan Aerospace Exploration Agency–Granada Crys-tallization Facility High Quality Protein Crystallization Project on the ProteinStructure and Function Analysis for Application (A.N.), and by the NationalProject on Protein Structural and Functional Analyses from the Ministry ofEducation, Culture, Sports, Science and Technology, Japan (A.N.).

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