Document Detail


Regulation of DNA methylation using different tensions of double strands constructed in a defined DNA nanostructure.
MedLine Citation:
PMID:  20078043     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
A novel strategy for regulation of an enzymatic DNA modification reaction has been developed by employing a designed nanoscale DNA scaffold. DNA modification using enzymes often requires bending of specific DNA strands to facilitate the reaction. The DNA methylation enzyme EcoRI methyltransferase (M.EcoRI) bends double helix DNA by 55 degrees-59 degrees during the reaction with flipping out of the second adenine in the GAATTC sequence as the methyl transfer reaction proceeds. In this study, two different double helical tensions, tense and relaxed states of double helices, were created to control the methyl transfer reaction of M.EcoRI and examine the structural effect on the methylation. We designed and prepared a two-dimensional (2D) DNA scaffold named the "DNA frame" using the DNA origami method that accommodates two different lengths of the double-strand DNA fragments, a tense 64mer double strand and a relaxed 74mer double strand. Fast-scanning atomic force microscope (AFM) imaging revealed the different dynamic movement of the double-strand DNAs and complexes of M.EcoRI with 64mer and 74mer double-strand DNAs. After treatment of the double strands in the DNA frame with M.EcoRI and the subsequent digestion with restriction enzyme EcoRI (R.EcoRI), AFM analysis revealed that the 74mer double-strand DNA was not effectively cleaved compared with the 64mer double-strand DNA, indicating that the methylation preferentially occurred in the relaxed 74mer double-strand DNA compared with that in the tense 64mer double strand. Biochemical analysis of the methylation and specific digestion using a real-time PCR also supported the above results. These results indicate the importance of the structural flexibility for bending of the duplex DNA during the methyl transfer reaction with M.EcoRI. Therefore, the DNA methylation can be regulated using the structurally controlled double-strand DNAs constructed in the DNA frame nanostructure.
Authors:
Masayuki Endo; Yousuke Katsuda; Kumi Hidaka; Hiroshi Sugiyama
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Journal of the American Chemical Society     Volume:  132     ISSN:  1520-5126     ISO Abbreviation:  J. Am. Chem. Soc.     Publication Date:  2010 Feb 
Date Detail:
Created Date:  2010-02-03     Completed Date:  2010-03-12     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  7503056     Medline TA:  J Am Chem Soc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  1592-7     Citation Subset:  IM    
Affiliation:
Institute for Integrated Cell-Material Sciences, Kyoto University, Yoshida-ushinomiyacho, Sakyo-ku, Kyoto 606-8501, Japan. endo@kuchem.kyoto-u.ac.jp
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MeSH Terms
Descriptor/Qualifier:
DNA / chemistry,  metabolism*
DNA Methylation*
Deoxyribonuclease EcoRI / metabolism
Methylation
Nanostructures / chemistry*,  ultrastructure
Nucleic Acid Conformation
Protein Binding
Site-Specific DNA-Methyltransferase (Adenine-Specific) / metabolism
Surface Properties
Chemical
Reg. No./Substance:
9007-49-2/DNA; EC 2.1.1.-/DNA modification methylase EcoRI; EC 2.1.1.72/Site-Specific DNA-Methyltransferase (Adenine-Specific); EC 3.1.21.-/Deoxyribonuclease EcoRI

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