Human Set and Ring associated (SRA) domain of UHRF1 bound to methylated DNA

Target ID:

UHRF1

Aliases:

FLJ21925hNP95ICBP90MGC138707Np95RNF106

Deposition Date:

Thursday 20th March 2008

Families:

Ubiquitin Related Biology

Related Diseases:

CancerChromatin and epigeneticsSignalling

Structure type:

protein-ligand

Download Coordinates:

3CLZ

Authors:

Walker JR, Avvakumov GV, Xue S, Dong A, Bountra C, Weigelt J, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

3CLZ

tabs

Structure Details

Epigenetic inheritance in mammals is characterized by high-fidelity replication of CpG methylation patterns during development1,2. UHRF1 (also known as ICBP90 in humans and Np95 in mouse)3 is an E3 ligase important for the maintainance of global and local DNA methylation in vivo4,5. It's preferential affinity for hemi-methylated over symmetrically-methylated DNA via its Set and Ring Associated (SRA) domain6 and its association with the maintenance DNA methyltransferase 1 (DNMT1) suggests a role in replication of the epigenetic code4,5,7. Here we report the 1.7 Å crystal structure of the apo SRA domain of human UHRF1 and a 2.2 Å structure of its complex with hemi-methylated DNA, revealing a novel reading mechanism for methylated CpG sites. The SRA-DNA complex has several dramatic structural features including a binding pocket that accommodates the 5-methyl-cytosine which is flipped out of the duplex DNA. Two specialized loops reach through the resulting gap in the DNA from both the major and minor grooves to read the other three bases of the CpG duplex. The major groove loop confers both specificity for the CpG dinucleotide and discrimination against methylation of deoxycytidine of the complementary strand. The structure, along with mutagenesis data, suggests how UHRF1 acts as a key factor for DNMT1 maintenance methylation through recognition of a fundamental unit of epigenetic inheritance, mCpG.

Materials and Methods

UHRF1

PDB:3CLZ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_037414
Entry Clone Source:ubh12.BC113875.OBS.MHS4426-98361361.pCR-BluntIITOPO
SGC Clone Accession:ubh12.414.617.125B05 (SDC125B05)
Tag:N-terminal: M
C-terminal: AHHHHHH
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mPSNHYGPIPGIPVGTMWRFRVQVSESGVHRPHVAGIHGRSNDGAYSLVLAGGYEDDVDHGNFFTYTGSGGRDLSGNKRTAEQSCDQKLTNTNRALALNCFAPINDQEGAEAKDWRSGKPVRVVRNVKGGKNSKYAPAEGNRYDGIYKVVKYWPEKGKSGFLVWRYLLRRDDDEPGPWTKEGKDRIKKLGLTMQYPEGYLEALANahhhhhh
Vector:pNIC-CH

Growth

Medium:TB
Antibiotics:
Procedure:The protein was expressed in E. coli BL21 (DE3) grown in Terrific Broth (TB) in the presence of 50 µg/ml of kanamycin at 37ºC to an OD600 of 7.5. Protein expression was induced 0.05 mM isopropyl-1-thio-D-galactopyranoside (IPTG) overnight at 15ºC. The culture was centrifuged (12,000 x g, 15 minutes) and cell pellets collected and stored at -80ºC.

Purification

Procedure
The cleared lysate was loaded onto a 3 mL TALON metal-affinity resin column (BD Biosciences) at 4ºC. The column was washed with 10 mL Wash buffer A, 10 mL Wash buffer B, and 10 mL Wash buffer A. The protein was eluted with 6 mL Elution buffer.

The protein was further purified by gel filtration on a HighLoad 16/60 Superdex 200 column (GE Healthcare, Amersham) equilibrated with Gel Filtration buffer. Fractions containing protein (analyzed by SDS-PAGE) were pooled and concentrated by ultrafiltration using an Amicon Ultra centrifugal filter with 10 kD cutoff to a final concentration of 34 mg/ml.

The yield of the protein was 4 mg per liter of bacterial culture.

Extraction

Procedure
The cell pellet from a 2 L culture was resuspended in 50 ml Lysis buffer, lysed using a Microfluidizer at 18,000 PSI, and cleared by centrifugation at 40,000 x g for 30 min.
Concentration:34 mg/ml
Ligand
MassSpec:Mass-spectroscopy by LC/MS showed pure product of correct molecular weight corresponding the UHRF1 SRA domain.
Crystallization:The protein at 10 mg/ml was mixed, in a molar ratio of 1:1.5, with a double-stranded DNA obtained by annealing of two oligonucleotides, 5Â-GGGCCXGCAGGG (X = 5-methylcytosine) and 5Â-CCCTGCGGGCCC synthesized by Oligos Etc., and incubated for 1 h on ice. Crystals of the complex were grown at 298 K using the hanging drop method by mixing 1 volume of the complex solution with 1 volume of well solution, consisting of 12% PEG1500, 0.2 M NaCl, 1 mM TCEP, 0.1 M bis-Tris, pH 7, and 0.4 volume of 30% xylitol. The crystals were cryoprotected by immersion in the well solution mixed in 1:1 ratio with a water solution containing 20% (w/v) sucrose, 4% (w/v) glucose, 18% (v/v) glycerol and 18% (v/v) ethylene glycol.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

Holliday, R. & Pugh, J. E. DNA modification mechanisms and gene activity during development. Science 187, 226-232 (1975).
Razin, A. & Riggs, A. D. DNA methylation and gene function. Science 210, 604-610 (1980).
Unoki, M., Bronner, C. & Mousli, M. A concern regarding the current confusion with the human homolog of mouse Np95, ICBP90/UHRF1. Radiat Res 169, 240-244 (2008).
Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908-912 (2007).
Bostick, M. et al. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 317, 1760-1764 (2007).
Unoki, M., Nishidate, T. & Nakamura, Y. ICBP90, an E2F-1 target, recruits HDAC1 and binds to methyl-CpG through its SRA domain. Oncogene 23, 7601-7610 (2004).
Achour, M. et al. The interaction of the SRA domain of ICBP90 with a novel domain of DNMT1 is involved in the regulation of VEGF gene expression. Oncogene 27, 2187-2197 (2008).