The
protein MeCP2 mediates epigenetic regulation by binding methyl-CpG
(mCpG) sites on chromatin. MeCP2 consists of six domains of which
one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA.
We show that solution conditions with physiological or greater salt
concentrations or the presence of nonspecific competitor DNA is necessary
for the MBD to discriminate mCpG from CpG with high specificity. The
specificity for mCpG over CpG is >100-fold under these solution
conditions.
In contrast, the MBD does not discriminate hydroxymethyl-CpG from
CpG. The MBD is unusual among site-specific DNA binding proteins in
that (i) specificity is not conferred by the enhanced affinity for
the specific site but rather by suppression of its affinity for generic
DNA, (ii) its specific binding to mCpG is highly electrostatic, and
(iii) it takes up as well as displaces monovalent cations upon DNA
binding. The MBD displays an unusually high affinity for single-stranded
DNA independent of modification or sequence. In addition, the MBD
forms a discrete dimer on DNA via a noncooperative binding pathway.
Because the affinity of the second monomer is 1 order of magnitude
greater than that of nonspecific binding, the MBD dimer is a unique
molecular complex. The significance of these results in the context
of neuronal function and development and MeCP2-related developmental
disorders such as Rett syndrome is discussed.
Although atomic-resolution crystal structures of the conserved C-terminal domain of several species of TBP and their complexes with DNA have been determined, little information is available concerning the structure in solution of full-length TBP containing both the conserved C-terminal and nonconserved N-terminal domains. Quantitation of the amino acid side chain oxidation products generated by synchrotron X-ray radiolysis by mass spectrometry has been used to determine the solvent accessibility of individual residues in monomeric Saccharomyces cerevisiae TATA binding protein (TBP) free in solution and in the TBP-DNA complex. Amino acid side chains within the C-terminal domain of unliganded full-length TBP that are predicted to be accessible from crystal structures of the isolated domain are protected from oxidation. Residues within the N-terminal domain are also protected from oxidation in both the absence and presence of DNA. Some residues within the DNA-binding "saddle" of the C-terminal domain are protected upon formation of a TBP-DNA complex as expected, while others are protected in both the absence and presence of bound DNA. In addition, residues on the upper side of the beta-sheets undergo reactivity changes as a function of DNA binding. These data suggest that the DNA-binding saddle of monomeric unliganded yeast TBP is only partially accessible to solvent, the N-terminal domain is partially structured, and the N- and C-terminal domains form a different set of contacts in the free and DNA-bound protein. The functional implications of these results are discussed.
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