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Title: The structure of the methyl-binding domain of MBD2, and studies of the MBD:DNA interactions
Author: Lillie, Kirsten L.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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DNA methylation is a powerful epigenetic control mechanism, essential for the regulation of transcriptional processes. In mammals, methylation occurs mainly at CpG dinucleotides and the modified sequence is recognised by members of the methylated DNA-binding domain (MBD) family. Methylated DNA binding domain 2 (MBD2) is a 44 kDa protein with the ability to distinguish between methylated and unmethylated DNA. It is associated with histone deacetylases and nucleosome remodelling complexes in vivo, and functions as a transcriptional repressor. MBD2 expression levels correlate with certain types of cancer and MBD2 may contribute to tumour development, by inappropriate silencing of tumour suppressor genes. MBD2 is a link between DNA methylation, chromatin structure and certain disease mechanisms. Understanding how methylated DNA is recognised by MBD2, will help establish its specific role as a methylation interpreter. This thesis describes the structure determination of the 72 residue MBD from mouse MBD2, using solution NMR spectroscopy. The MBD has a characteristic wedge-shaped fold, with one face made up of 13-sheets, and the other of an a-helix plus a hairpin loop. Loop Li, between two long beta-strands, is much more rigid in this structure compared to the equivalent region in other members of the MBD family. It appears to be a pre-formed DNA binding loop and overlays well with the structure of the MBD (from MBD1):DNA complex, solved by another group. NMR was subsequently used to follow the titration of the MBD from MBD2, with a 12 base-pair DNA oligomer, symmetrically methylated at a central CpG pair. Large chemical shift changes in the '5N-HSQC spectrum were observed for Li loop residues, and for an arginine residue at the base of the a-helix. This corresponds to the DNA binding interface in the MBD1 MBD:DNA complex structure. Surface plasmon resonance was used to compare the affinities of three different MBDs for DNA oligomers in various methylation states. The MBD2 MBD showed weaker binding to symmetrically methylated DNA, when compared to MeCP2 and MBD4 MBDs. In general, there was no difference in affinity for any of the methylated oligomers, and no construct bound unmethylated DNA. There is some evidence that oligomer length affects binding, although this was not studied in detail.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available