Protein design of the mammalian DNA methyltransferase Dnmt3a

  • The catalytic domain of the Dnmt3a DNA-(cytosine C5)-methyltransferase (Dnmt3a-C) is active in isolated form but like full-length Dnmt3a it shows only weak DNA methylation activity. To improve this activity by directed evolution, we set up a selection system in which Dnmt3a-C methylate its own expression plasmid in E. coli and protect it from cleavage with methylation specific restriction enzymes. However, despite screening about 400 clones which were selected in 3 rounds from a library of 60000 clones, we were not able to isolate a variant with improved activity, most likely because of a background of uncleaved plasmids and plasmids which have lost the restriction site. We also showed that the catalytic domain of mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-L-methionine (AdoMet) to a cysteine residue in its catalytic centre. This reaction is reversible and slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a AdoMet complexes. In the presence of CpG containing double stranded DNA, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. This reaction might constitute a regulatory mechanism which could inactivate unused Dnmt3a in the cell.

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Publishing Institution:IRC-Library, Information Resource Center der Jacobs University Bremen
Granting Institution:Jacobs Univ.
Author:Abu Nasar Siddique
Referee:Albert Jeltsch, Sebastian Springer, Maranne G. Rots
Advisor:Albert Jeltsch
Persistent Identifier (URN):urn:nbn:de:101:1-2013052812468
Document Type:PhD Thesis
Language:English
Date of Successful Oral Defense:2011/05/25
Date of First Publication:2011/05/30
PhD Degree:Biotechnology
School:SES School of Engineering and Science
Library of Congress Classification:Q Science / QH Natural history - Biology / QH301-705.5 Biology (General) / QH426-470 Genetics / QH442 Molecular genetics. Genetic engineering
Call No:Thesis 2011/19

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