Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

Abstract

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G · T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.

Keywords: 5-Methylcytosine; Base excision repair; CpG site; DNA glycosylase; Deamination; Demethylation; G·T mispair; Methyl binding domain 4; Oxidation; Thymine DNA glycosylase.

Fig. 1

Pathway for active DNA demethylation mediated by TET enzymes followed by TDG-initiated BER. TET enzymes (noted in red) catalyze stepwise oxidation of 5mC to 5hmC, 5fC, and 5caC. TDG excises 5fC and 5caC and subsequent BER steps yield cytosine.

Fig. 2

Structures of MBD4. (A) Schematic primary structure of MBD4. The MBD and the glycosylase domain are structured, while other regions appear to be disordered. (B) Crystal structure of the MBD bound to a G·T mispair in a CpG context, i.e., 5mCG/TG (PDBID: 3VXV). The DNA is shown as gray lines with surface representation, the G·T mispair is shown in cyan and the flanking C·G pair (CpG context) is in yellow (with N and O atoms blue and red). The same coloring scheme is used in panels C, D, and E. Note that the structures in panels B through E are oriented in the same way with respect to the position of the mismatched guanine (cyan, with an asterisk).(C) Structure of the MBD4 glycosylase domain bound to DNA, with thymine of a G·T mispair flipped into the active site (PDBID: 4E9G). (D) Close up view of the MBD (as in panel B) shows that it recognizes an 5mCG/GT dinucleotide using conserved Arg and other residues together with water molecules. (E) Close-up view of contacts the glycosylase forms with the flipped thymine, via Tyr and Gln side chains and backbone contacts. Also shown are contacts with mismatched G, and the Arg side chain that fills the void created by thymine flipping.

Fig. 3

Structures and molecular dynamics (MD) studies of human TDG. (A) Schematic primary structure of TDG showing the catalytic (glycosylase) domain and the two flanking regions that are disordered yet functionally important. Shown are sites of post-translational modifications, the SUMO-interacting motif (SIM), and PIP degron that mediates interaction with PCNA and subsequent ubiquitination and degradation. (B) Structure of TDG (catalytic domain) bound to DNA with U of a G·U mispair flipped into active site (2.97 Å resolution, PDBID: 3UFJ). The DNA is shown as gray lines with surface representation; the G·U mispair is shown in cyan and the flanking C·G pair (i.e., CpG context) is yellow. Contacts with the flipped U and mismatched G are shown. (C) Structure of SUMO-1 modified TDG (residues 117–331, PDBID: 1WYW), with DNA modeled in (by aligning DNA-bound TDG; PDBID 2RBA). The SUMO-1 domain is covalently tethered to K330 of TDG and it forms many non-covalent interactions with the SIM of TDG (left surface as shown). SUMO-binding stabilizes an otherwise disordered helix of TDG (magenta), which likely weakens TDG binding to AP-DNA. (D) To illustrate a potential steric clash involving the methyl groups of a flipped thymine and Ala145, uracil from the structure in panel B was replaced with thymine (interactions shown are from the structure in panel B). (E) MD simulations of TDG bound to a G·T mispair suggest that the enzyme can transiently flip thymine into a position that lies just shy of the fully-flipped conformation which is likely needed for base excision. The last two panels are adapted from Ref [60].