Skip Navigation

Nucleic Acids Research 2005 33(22):7039-7047; doi:10.1093/nar/gki1009
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (873K) Freely available
Right arrow Screen PDF (291K) 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 (9)
Right arrow Commercial Re-use Guidelines
for Open Access NAR Content
Google Scholar
Right arrow Articles by Eisenschmidt, K.
Right arrow Articles by Pingoud, A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Eisenschmidt, K.
Right arrow Articles by Pingoud, A.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 14 December 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

Article

Developing a programmed restriction endonuclease for highly specific DNA cleavage

Kristin Eisenschmidt, Thomas Lanio, András Simoncsits1, Albert Jeltsch2, Vera Pingoud, Wolfgang Wende and Alfred Pingoud*

Institut für Biochemie, Justus-Liebig-Universität Heinrich-Buff-Ring 58, D-35392 Giessen, Germany 1International Centre for Genetic Engineering and Biotechnology Padriciano 99, I-34012 Trieste, Italy 2School of Engineering and Science, International University Bremen Campus Ring 1, D-28725 Bremen, Germany

*To whom correspondence should be addressed. Tel: +49 641 9935400; Fax: +49 641 9935409; Email: alfred.m.pingoud{at}chemie.bio.uni-giessen.de

Received November 1, 2005. Revised November 24, 2005. Accepted November 24, 2005.

Specific cleavage of large DNA molecules at few sites, necessary for the analysis of genomic DNA or for targeting individual genes in complex genomes, requires endonucleases of extremely high specificity. Restriction endonucleases (REase) that recognize DNA sequences of 4–8 bp are not sufficiently specific for this purpose. In principle, the specificity of REases can be extended by fusion to sequence recognition modules, e.g. specific DNA-binding domains or triple-helix forming oligonucleotides (TFO). We have chosen to extend the specificity of REases using TFOs, given the combinatorial flexibility this fusion offers in addressing a short, yet precisely recognized restriction site next to a defined triple-helix forming site (TFS). We demonstrate here that the single chain variant of PvuII (scPvuII) covalently coupled via the bifunctional cross-linker N-({gamma}-maleimidobutryloxy) succinimide ester to a TFO (5'-NH2-[CH2]6 or 12-MPMPMPMPMPPPPPPT-3', with M being 5-methyl-2'-deoxycytidine and P being 5-[1-propynyl]-2'-deoxyuridine), cleaves DNA specifically at the recognition site of PvuII (CAGCTG) if located in a distance of approximately one helical turn to a TFS (underlined) complementary to the TFO (‘addressed’ site: 5'-TTTTTTTCTCTCTCTCN~10CAGCTG-3'), leaving ‘unaddressed’ PvuII sites intact. The preference for cleavage of an ‘addressed’ compared to an ‘unaddressed’ site is >1000-fold, if the cleavage reaction is initiated by addition of Mg2+ ions after preincubation of scPvuII-TFO and substrate in the absence of Mg2+ ions to allow triple-helix formation before DNA cleavage. Single base pair substitutions in the TFS prevent addressed DNA cleavage by scPvuII-TFO.


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
S.-h. Chan, Y. Bao, E. Ciszak, S. Laget, and S.-y. Xu
Catalytic domain of restriction endonuclease BmrI as a cleavage module for engineering endonucleases with novel substrate specificities
Nucleic Acids Res., September 25, 2007; 35(18): 6238 - 6248.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
Y. Yamamoto, M. Mori, Y. Aiba, T. Tomita, W. Chen, J.-M. Zhou, A. Uehara, Y. Ren, Y. Kitamura, and M. Komiyama
Chemical modification of Ce(IV)/EDTA-based artificial restriction DNA cutter for versatile manipulation of double-stranded DNA
Nucleic Acids Res., April 1, 2007; 35(7): e53 - e53.
[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.