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Title: Studies of DNA demethylation
Author: Tsai, Hsin-hao
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2004
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Bacteriophage T3 has been reported to express S-adenosylmethionine hydrolase (SAMase) during the early stage of phage infection. In order to overcome the restriction-modification (R-M) system, SAMase destroys the modification cofactor S-adenosylmethionine (SAM) and hydrolyses SAM into homoserine and methylthioadenosine (MTA). Since SAM is also the major donor of the methyl groups incorporated in DNA methylation, this SAM cleaving activity may be utilized as a demethylating agent, which helps us to understand the mechanism of DNA demethylation. However, the reaction mechanism of T3 SAMase has not been very well studied. To further elucidate this mechanism, experiments were carried out to purify recombinant SAMase and to enable attempts to solve the crystal structure of this enzyme. In addition, we aimed to observe effects of DNA demethylation in mammalian cells by using this hydrolase activity to reduce the cellular level of SAM. SAMase was also chosen to substitute the more commonly used demethylating agent, 5-azaC, in order to avoid the highly toxic impact on drug-receiving cells. Additionally, RNA interference (RNAi), was utilised to explore the impact of DNA demethylation. Using small interfering RNA (siRNA), we depleted the mRNA of the enzyme responsible for maintenance methylation, DNA methyltransferase 1 (Dnmtl), and observed a correlation between DNA demethylation and Xist expression when the methyl-binding protein MBD2 was removed. Furthermore, the oxidative DNA repair has been suggested as a candidate pathway, which involves demethylation of the 1-methyladenine and 3-methylcytosine via a hydroxmethyl intermediate. To explore this possible mechanism, we investigated the candidate pathway by searching for a putative intermediate of hydroxymethyl cytosine during active DNA demethylation in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available