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Title: Investigation on the structure of CpG methylated DNA
Author: Reilly, Carmel M.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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It is known that CpG methylation can promote transitions between the A-, B- and Z- DNA isoforms. DNA methylation has also been shown to have effects on both secondary and tertiary DNA structure. These alterations can affect the positioning of the primary DNA packaging proteins in vitro and potentially, higher order chromatin structure. A variety of methyl-CpG binding proteins (MeCPs) have now been identified. These in turn interact with a variety of chromatin associated proteins capable of interpreting and reacting to the information provided at methylated CpG sites leading to subsequent changes in gene expression. Here, I have investigated the structural effects of CpG methylation by a novel method. I have developed an assay that is able to detect CpG methylation induced conformational changes in symmetrically methylated double-stranded DNA. The assay is based on the enzymatic properties of Benzonase, a nuclease that preferentially digests A-form DNA in vitro. In this study, a range of DNA gene fragments were used as Benzonase digestion substrates to look for potential A-form regions in CpG methylated DNA. The results suggest that in certain sequence contexts, CpG methylation promotes the formation of A-DNA. These regions of potential A-form DNA are also coincident with sequence motifs that have crystallised as A-form DNA. In order to test the biological relevance of these results, the affinity of specially designed methylated and unmethylated substrates for a variety of methyl-binding protein (MeCP2, Kaiso, xMBD1 and xMBD3) was investigated. The results of these experiments are discussed in the text. From these results, I hypothesise that above the layer of information provided by the DNA sequence, is another layer of coded information provided by DNA methylation. This information is represented by 3-dimensional alterations at both secondary and tertiary structural levels of the DNA helix. These alterations may be in turn, recognised by an array of MeCPs, which in turn interact with proteins which can cause an array of modifications to the nucleosomal histone tails. This tailored display on the histone tails constitutes another set of coded information to influence higher order chromatin proteins, which will operate co-ordinately to effect subsequent localised and/or cellular functional consequences.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available