Solution structure and intramolecular exchange of methyl-cytosine binding domain protein 4 (MBD4) on DNA suggests a mechanism to scan for mCpG/TpG mismatches

Abstract

Unlike other members of the methyl-cytosine binding domain (MBD) family, MBD4 serves as a potent DNA glycosylase in DNA mismatch repair specifically targeting mCpG/TpG mismatches arising from spontaneous deamination of methyl-cytosine. The protein contains an N-terminal MBD (MBD4MBD) and a C-terminal glycosylase domain (MBD4GD) separated by a long linker. This arrangement suggests that the MBD4MBD either directly augments enzymatic catalysis by the MBD4GD or targets the protein to regions enriched for mCpG/TpG mismatches. Here we present structural and dynamic studies of MBD4MBD bound to dsDNA. We show that MBD4MBD binds with a modest preference formCpG as compared to mismatch, unmethylated and hydroxymethylated DNA. We find that while MBD4MBD exhibits slow exchange between molecules of DNA (intermolecular exchange), the domain exhibits fast exchange between two sites in the same molecule of dsDNA (intramolecular exchange). Introducing a single-strand defect between binding sites does not greatly reduce the intramolecular exchange rate, consistent with a local hopping mechanism for moving along the DNA. These results support a model in which the MBD4MBD4 targets the intact protein to mCpG islands and promotes scanning by rapidly exchanging between successive mCpG sites which facilitates repair of nearby mCpG/TpG mismatches by the glycosylase domain.

Structural Basis of MeCP2 Distribution on Non-CpG Methylated and Hydroxymethylated DNA

“…The residue-specific changes in the amide backbone of MeCP2 bound to the BDNF promoter sequences were monitored by 1 H/ 15 N-Heteronuclear Single-Quantum Correlation (HSQC) NMR spectroscopy and compared using standard chemical shift perturbations (CSPs). In NMR studies of the MBD family proteins, we identified linear chemical shift changes that correlate with methylated DNA binding and present in every ortholog and paralog examined to date [13,26–28]. In particular, two of these resonances (Gly 27 and Ala 30 of MBD2) show large CSPs between the non-specific and methylation-specific binding modes, such that we have used these resonances as reporters of mCG binding [27].…”

“…As we have described previously for MBD4 [28], MeCP2 has a small insertion of 4 amino acids that extends helix 1 by one additional turn as compared to MBD2. This addition contributes to a larger hydrophobic core possibly stabilizing the isolated domain.…”

Methylation specific targeting of a chromatin remodeling complex from sponges to humans

“…The methylated DNA sequence was generated with the 3D-DART webserver and subsequent molecular dynamics simulations were performed essentially as described previously15 using NAMD 2.943 and CHARMM27 force field44. In brief, the initial complexes were solvated in a box with at least 10 Å of surrounding TIP3P water and 75 mM NaCl and equilibrated with two rounds of NVT simulations (5000 steps of minimization followed 30 ps of dynamics at 300 K) with rigid (first round) or 5 kcal/mol/Å harmonic (second round) restraints.…”

“…Our laboratory has been studying the structure and function of the methyl-cytosine binding domain (MBD) family of proteins12131415. These proteins share an approximately 60 amino acid domain that can selectively bind to symmetrically methylated CpG dinucleotides.…”

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

“…However, as noted previously [63], and shown in Figures 2B and 2C, the sharp (57°) bend in DNA imposed by the glycosylase domain[74–76]seems likely to disrupt binding of the MBD, which binds straight DNA [65]. Taken together, findings to date indicate that the two domains bind unique sites of DNA, and that the MBD targets full-length MBD4 to regions of methylated CpG sites so that the glycosylase domain can more efficiently find deaminated 5mC bases [55, 56, 63, 83, 84]. The 280-residue linker joining the domains, which is likely disordered, could allow up to 50 bp separation between binding sites for the two domains [83].…”