Skip Navigation

Nucleic Acids Research 2004 32(13):3898-3903; doi:10.1093/nar/gkh724
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (125K) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (4)
Right arrowRequest Permissions
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Tímár, E.
Right arrow Articles by Venetianer, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Tímár, E.
Right arrow Articles by Venetianer, P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 25 July 2004

Nucleic Acids Research, Vol. 32 No. 13 © Oxford University Press 2004; all rights reserved

Changing the recognition specificity of a DNA-methyltransferase by in vitro evolution

Edit Tímár, Gergely Groma, Antal Kiss and Pál Venetianer*

Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, Szeged 6701, Hungary

* To whom correspondence should be addressed. Tel: +36 62 599 650; Fax: +36 62 433 506; Email: venetianer{at}brc.hu

Received May 27, 2004; Revised and Accepted July 9, 2004

The gene coding for the SinI DNA-methyltransferase, a modification enzyme able to recognize and methylate the internal cytosine of the GGA/TCC sequence, was subjected to in vitro mutagenesis, DNA-shuffling and a strong selection for relaxed GGNCC recognition specificity. As a result of this in vitro evolution experiment, a mutant gene with the required phenotype was selected. The mutant SinI methyltransferase carried five amino acid substitutions. None of these was found in the ‘variable region’ that were thought to be responsible for sequence specificity. Three were located near the N-terminal end, preceding the first conserved structural motif of the enzyme; two were found between conserved motifs VI and VII. A clone engineered to carry out only the latter two replacements (L214S and Y229H) displays relaxed recognition specificity similar to that of the parental mutant, whereas the clone carrying only the N-terminal replacements showed a much weaker change in recognition specificity. The enzyme with two internal mutations was purified and characterized. Its catalytic activity (kcat/Km) was ~5-fold lower towards GGA/TCC and 20-fold higher towards GGG/CCC than that of the wild-type enzyme.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Nucleic Acids ResHome page
R. Gerasimaite, G. Vilkaitis, and S. Klimasauskas
A directed evolution design of a GCG-specific DNA hemimethylase
Nucleic Acids Res., October 8, 2009; (2009) gkp772v2.
[Abstract] [Full Text] [PDF]

Home page
 J. Bacteriol.Home page
E. Timar, P. Venetianer, and A. Kiss
In Vivo DNA Protection by Relaxed-Specificity SinI DNA Methyltransferase Variants
J. Bacteriol., December 15, 2008; 190(24): 8003 - 8008.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
F. Zhao, X. Zhang, C. Liang, J. Wu, Q. Bao, and S. Qin
Genome-wide analysis of restriction-modification system in unicellular and filamentous cyanobacteria
Physiol Genomics, February 23, 2006; 24(3): 181 - 190.
[Abstract] [Full Text] [PDF]


Disclaimer: Please note that abstracts for content published before 1996 were created through digital scanning and may therefore not exactly replicate the text of the original print issues. All efforts have been made to ensure accuracy, but the Publisher will not be held responsible for any remaining inaccuracies. If you require any further clarification, please contact our Customer Services Department.