Skip Navigation

Nucleic Acids Research 2005 33(2):661-671; doi:10.1093/nar/gki213
This Article
Right arrow Full Text Freely available
Right arrow Print PDF (2896K) Freely available
Right arrow Screen PDF (591K) Freely available
Right arrow Supplementary Material
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 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 Pawlak, S. D.
Right arrow Articles by Skowronek, K. J.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Pawlak, S. D.
Right arrow Articles by Skowronek, K. J.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Published online 31 January 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}oupjournals.org.

Article

Inference of relationships in the ‘twilight zone’ of homology using a combination of bioinformatics and site-directed mutagenesis: a case study of restriction endonucleases Bsp6I and PvuII

Sebastian D. Pawlak, Monika Radlinska1, Agnieszka A. Chmiel, Janusz M. Bujnicki* and Krzysztof J. Skowronek

Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology ul. ks. Trojdena 4, 02-109 Warsaw, Poland 1 Institute of Microbiology, Warsaw University ul. Miecznikowa 1, 02-096 Warsaw, Poland

*To whom correspondence should be addressed. Tel: +48 22 668 5384; Fax: +48 22 668 5288; Email: iamb{at}genesilico.pl

Received November 3, 2004. Revised January 7, 2005. Accepted January 7, 2005.

Thus far, identification of functionally important residues in Type II restriction endonucleases (REases) has been difficult using conventional methods. Even though known REase structures share a fold and marginally recognizable active site, the overall sequence similarities are statistically insignificant, unless compared among proteins that recognize identical or very similar sequences. Bsp6I is a Type II REase, which recognizes the palindromic DNA sequence 5'GCNGC and cleaves between the cytosine and the unspecified nucleotide in both strands, generating a double-strand break with 5'-protruding single nucleotides. There are no solved structures of REases that recognize similar DNA targets or generate cleavage products with similar characteristics. In straightforward comparisons, the Bsp6I sequence shows no significant similarity to REases with known structures. However, using a fold-recognition approach, we have identified a remote relationship between Bsp6I and the structure of PvuII. Starting from the sequence–structure alignment between Bsp6I and PvuII, we constructed a homology model of Bsp6I and used it to predict functionally significant regions in Bsp6I. The homology model was supported by site-directed mutagenesis of residues predicted to be important for dimerization, DNA binding and catalysis. Completing the picture of sequence–structure–function relationships in protein superfamilies becomes an essential task in the age of structural genomics and our study may serve as a paradigm for future analyses of superfamilies comprising strongly diverged members with little or no sequence similarity.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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. D. Morgan, E. A. Dwinell, T. K. Bhatia, E. M. Lang, and Y. A. Luyten
The MmeI family: type II restriction-modification enzymes that employ single-strand modification for host protection
Nucleic Acids Res., July 3, 2009; (2009) gkp534v1.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Orlowski and J. M. Bujnicki
Structural and evolutionary classification of Type II restriction enzymes based on theoretical and experimental analyses
Nucleic Acids Res., June 1, 2008; 36(11): 3552 - 3569.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
M. Y. Niv, D. R. Ripoll, J. A. Vila, A. Liwo, E. S. Vanamee, A. K. Aggarwal, H. Weinstein, and H. A. Scheraga
Topology of Type II REases revisited; structural classes and the common conserved core
Nucleic Acids Res., April 1, 2007; 35(7): 2227 - 2237.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
E. Armalyte, J. M. Bujnicki, J. Giedriene, G. Gasiunas, J. Kosinski, and A. Lubys
Mva1269I: A Monomeric Type IIS Restriction Endonuclease from Micrococcus Varians with Two EcoRI- and FokI-like Catalytic Domains
J. Biol. Chem., December 16, 2005; 280(50): 41584 - 41594.
[Abstract] [Full Text] [PDF]

Home page
 Nucleic Acids ResHome page
J. Soding, A. Biegert, and A. N. Lupas
The HHpred interactive server for protein homology detection and structure prediction
Nucleic Acids Res., July 1, 2005; 33(suppl_2): W244 - W248.
[Abstract] [Full Text] [PDF]

Home page
 Protein Eng Des SelHome page
A. A. Chmiel, M. Radlinska, S. D. Pawlak, D. Krowarsch, J. M. Bujnicki, and K. J. Skowronek
A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy
Protein Eng. Des. Sel., April 1, 2005; 18(4): 181 - 189.
[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.