Fig 1.
Single particle cryo-EM reconstruction of apo DNMT1.
(A) Domain organization of human DNMT1. (B) Representative 2D class averages calculated from micrographs showing different particle orientations. (C) Cryo-EM density map of apo DNMT1 in front and side views. Domains are color-coded as in (A). (D) Cryo-EM density map colored according to the resolution distribution at higher (upper panel) and lower (lower panel) contour levels. The resolution (Å) is color-coded as indicated on the bar. (E) Representative regions of the cryo-EM map with the crystal structure of apo human DNMT1 (PDB 4WXX) fitted in.
Table 1.
Cryo-EM data collection of apo DNMT1, DNMT1: Non-productive DNA complex, DNMT1: H3Ub2-peptide, DNMT1: Productive DNA complex with refinement statistics for apo DNMT1.
Fig 2.
DNMT1 interaction with hemi-methylated DNA.
(A) Binding of DNMT1 to DNA in the presence and absence of GSK3852279B inhibitor (labeled as [C]) analysed by native gel electrophoresis. The gel is stained to detect either protein (left) or DNA (right). (B) Cryo-EM density map and 2D analysis of the DNMT1: DNA: GSK3852279B complex showing the non-productive state. The DNMT1 domains are colored as in Fig 1A. The DNA density is indicated by yellow arrows (2D) and colored in orange (3D). Crystal structure PDB 3PTA [34] has been placed into the cryo-EM density. (C) DNA methylation by DNMT1 in the presence of H3Ub2-peptide and GSK3852279B inhibitor (labeled as [C]) analyzed by SDS-PAGE. The gel is stained to detect either protein (left) or DNA (right). Free DNMT1 and DNMT1 cross-linked to 5FDNA (DNMT1-5FDNA) are indicated. (D) Cryo-EM density map and 2D analysis of the DNMT1:5FDNA complex in the presence of H3Ub2-peptide showing the productive state. The DNMT1 domains are colored as in Fig 1A. The DNA density is indicated by yellow arrows (2D) and colored in orange (3D). Crystal structure PDB 4DA4 [9] has been placed into the cryo-EM density.
Fig 3.
Cryo-EM map of full-length DNMT1 in complex with a di-ubiquitinated histone H3 peptide (H3Ub2-peptide).
(A) Cryo-EM density map showing DNMT1 (grey) and additional densities for the two ubiquitin molecules of the histone H3 peptide analogue (wheat) in front and side views. The sequence of the H3 peptide and the positions of Ub-18 and Ub-23 attached to it (red boxes) are indicated in the lower panel. (B) Superposition of the RFTS domain from apo DNMT1 (PDB ID 4WXX; RFTS in dark blue; other DNMT1 domains shown in grey surface) and RFTS from the RFTS-H3Ub2 crystal structure (PDB ID 5WVO; RFTS in light blue; ubiquitin molecules in grey cartoon). The bend in the α4-helix of RFTS is indicated by a red arrow and shown in the close-up view on the right. (C) Two alternative fits (on the left and right) of the RFTS: H3Ub2 crystal structure (PDB ID 5WVO, light blue RFTS, grey H3Ub2) into the cryo-EM density map of DNMT1: H3Ub2-peptide (lower panel). RFTS domain from the apo DNMT1 crystal structure (dark blue, upper panel) is provided as reference. (D) Fit of the apo DNMT1 structure (PDB ID 4WXX, domains colored as in Fig 1A) and the two ubiquitin molecules from the RFTS: H3Ub2 structure (PDB ID 5WVO) into the cryo-EM map of DNMT1: H3Ub2-peptide.
Fig 4.
Schematic diagram of the DNMT1 reaction cycle.
Apo DNMT1 is in an auto-inhibited state with the RFTS domain blocking access of the DNA to the catalytic domain (left panel). Binding of hemi-methylated DNA in a non-productive state and/or di-ubiquitinated histone H3 releases the autoinhibition of apo DNMT1 (middle panel). DNMT1 transitions into a productive state where hemi-methylated DNA can enter the active site to become fully methylated (right panel).