Solution structure of hMBD1 CXXC1.
PhD thesis, University of Glasgow.
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Methylation of CpG dinucleotides is the major epigenetic modification of mammalian DNA which results in the remodelling of transcriptionally active euchromatin to transcriptionally inactive heterochromatin. Recognition of methylated CpG by methylated DNA binding proteins, the MBD family, the Kaiso zinc finger family and the SRA domain proteins results in deacetylation and methylation of histone side chains through the recruitment of HDAC and HMT enzymes. Methylation of DNA is a heritable process ensuring Methylation dependant transcriptional repression is passed from mother to daughter cell during replication. Some of the proteins involved in this chromatin remodelling, MBD1, DNMT1, MLL, and CFP1 contain CXXC domains. hMBD1 contains 2 or 3 CXXC domains depending on the splice variant, with only the third CXXC domain shown to bind CpG dinucleotides.
This thesis describes the work done to elucidate the structure of hMBD1 CXXC1 and to investigate hMBD1 CXXC12 di-domain by NMR spectroscopy and biochemical characterisation. The hMBD1 CXXC1 & CXXC12 domains were successfully over expressed in E. coli and purified. Unlabelled and uniformly 15N labelled proteins were produced for nuclear magnetic resonance (NMR) studies. Assignment of NMR spectra was carried out and constraints generated enabling structure determination of hMBD1 CXXC1 and to investigate the relationship between CXXC1 and CXXC2 of hMBD1.
The solution structure of hMBD1 CXXC1 determined here was compared to the previously determined solution structure of hMLL CXXC in order to investigate their differences in DNA binding. NOE data from hMBD1 CXXC1 and CXXC12 are compared in order to investigate the domain structure of CXXC12. DNA titration of hMBD1 CXXC1 showed no significant interaction with a single CpG oligo while the loop region of hMBD1 CXXC1 differs significant in both structure and surface charge suggesting the loop region to be important for DNA binding. The recorded NOE data of hMBD CXXC12 suggests the two CXXC domains form a globular rather than a linear structure
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