Human DNA methyltransferase 1 associated protein 1, SANT domain

Target ID:

DMAP

Deposition Date:

Saturday 30th May 2009

Families:

Transferases

Structure type:

apo

Download Coordinates:

3HM5

Authors:

Dombrowski L, Tempe W, Amaya MF, Tong Y, Ni S, Bountra C, Weigelt J, Edwards AM, Arrowsmith CH, Bochkarev A, Min J, Park HW, Wu H

Site:

Toronto

3HM5

tabs

Structure Details

DNMT1-associated protein 1 (DMAP1) is a subunit of several, distinct complexes involved in the repression or activation of transcription. The protein can independently repress transcription and is targeted to replication foci throughout S phase by interacting directly with the N-terminus of DNA methyltransferase 1 (DNMT1). During late S phase, histone deacetylase 2 (HDAC2) is added to this complex, providing a means to deacetylate histones in transcriptionally inactive heterochromatin following replication. This protein is also a component of the nucleosome acetyltransferase of H4 complex (NuA4) and interacts with the transcriptional corepressor tumor susceptibility gene 101 and the pro-apoptotic death-associated protein 6.

DMAP1 protein contains a SANT domain at the N-terminus. SANT domain is a novel motif found in a number of eukaryotic transcriptional regulatory proteins. It was initially identified as a ~50 amino acids motif in nuclear receptor co-repressors, and subsequently found in the subunits of many chromatin remodeling complexes. The sequence of the SANT domains showed strong similarity to the DNA-binding domain of Myb-related proteins. Recent studies have indicated that the SANT domain plays a central role in chromatin remodeling by functioning as a unique histone-interaction module that couples histone binding to enzyme catalysis. We have solved the crystal structure of the SANT domain of DMAP1 protein at 1.8 Å resolution.

Materials and Methods

DMAP1

PDB:3HM5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_061973
Entry Clone Source:MGC
SGC Clone Accession:
Tag:N-terminal: His-tag with integrated TEV protease site: MHHHHHHSSGRENLYFQG
Host:E.coli BL21 (DE3) codon plus RIL (Stratagene).

Construct

Prelude:
Sequence:gGKDYPFARFNKTVQVPVYSEQEYQLYLHDDAWTKAETDHLFDLSRRFDLRFVVIHDRYDHQQFKKRSVEDLKERYYHICAKLANVRAVPGTD
Vector:pET28-MHL

Growth

Medium:
Antibiotics:
Procedure:The SANT domain of DMAP1 was expressed in E.coli BL21 (DE3) codon plus in Terrific Broth medium in the presence of 50 μg/ml of kanamycin. Cell were grown at 37 degC to an OD600 of 1.5 and induced by isopropyl-1-thio-D-galactopyranoside (IPTG), final concentration 1 mM and incubated overnight at 15 degC.

Purification

Procedure
The lysate was loaded onto 5 ml HiTrap column (Amersham Biosciences), charged with Ni2+. The column was washed with 10 CV of wash buffer, and the protein was eluted with elution buffer. The protein was then loaded on to a Superdex200 (60X60, Amersham Biosciences) column equilibrated in 20 mM PIPES, pH 6.5 buffer containing 250 mM NaCl. TEV protease was added to combined fractions containing DMAP1. The cut and uncut protein of DMAP1 were separated on a Ni column. The protein was further purified to homogeneity by ion-exchange chromatography on Source 30S column (10x10) (Amersham Biosciences), equilibrated with buffer 20 mM PIPES, pH 6.5, and eluted with linear gradient of NaCl up to 500 mM concentration (20CV). Purification yield was 4.5 mg of the protein per 1L of culture.

Extraction

Procedure
Cells were harvested by centrifugation at 7,000 rpm. The cell pellets were frozen in liquid nitrogen and stored at -80˚C. For the purification the cell paste was thawed and resuspended in lysis buffer with protease inhibitor (1mM phenylmethyl sulfonyl fluoride, PMSF). The cells were lysed by passing through Microfluidizer (Microfluidics Corp.) at 20,000 psi.
Concentration:11.5 mg/ml
Ligand
MassSpec:Expected MW is 11196.5 Da, measured mass is 11196.0 Da.
Crystallization:Purified SANT domain of DMAP1 was crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1 µl of the protein solution (10 mg/ml) with 1 µl of the reservoir solution containing 20% PEG 3350, 0.2 M CaCl2, 0.1 M HEPES, pH 7.0. Crystals were frozen in liquid nitrogen using glycerol as cryoprotectant.
NMR Spectroscopy:
Data Collection:
Data Processing: