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Nucleic Acids Research Advance Access published online on September 27, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn621
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic acid Enzymes

How PspGI, catalytic domain of EcoRII and Ecl18kI acquire specificities for different DNA targets

Gintautas Tamulaitis1, Mindaugas Zaremba1, Roman H. Szczepanowski2,3, Matthias Bochtler2,3,4 and Virginijus Siksnys1,*

1Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania, 2International Institute of Molecular and Cell Biology, Trojdena 4, 02-109 Warsaw, Poland, 3Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01309 Dresden, Germany and 4Schools of Chemistry and Biosciences, Main Building, Park Place, Cardiff University, Cardiff CF10 3AT, UK

*To whom correspondence should be addressed. Tel: +370 5 2602108; Fax: +370 5 2602116; Email: siksnys{at}ibt.lt

Received July 28, 2008. Revised September 11, 2008. Accepted September 11, 2008.

Restriction endonucleases Ecl18kI and PspGI/catalytic domain of EcoRII recognize CCNGG and CCWGG sequences (W stands for A or T), respectively. The enzymes are structurally similar, interact identically with the palindromic CC:GG parts of their recognition sequences and flip the nucleotides at their centers. Specificity for the central nucleotides could be influenced by the strength/stability of the base pair to be disrupted and/or by direct interactions of the enzymes with the flipped bases. Here, we address the importance of these contributions. We demonstrate that wt Ecl18kI cleaves oligoduplexes containing canonical, mismatched and abasic sites in the central position of its target sequence CCNGG with equal efficiencies. In contrast, substitutions in the binding pocket for the extrahelical base alter the Ecl18kI preference for the target site: the W61Y mutant prefers only certain mismatched substrates, and the W61A variant cuts exclusively at abasic sites, suggesting that pocket interactions play a major role in base discrimination. PspGI and catalytic domain of EcoRII probe the stability of the central base pair and the identity of the flipped bases in the pockets. This ‘double check’ mechanism explains their extraordinary specificity for an A/T pair in the flipping position.

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


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