Nucleic Acids Research Advance Access originally published online on September 8, 2009
Nucleic Acids Research 2009 37(20):6859-6870; doi:10.1093/nar/gkp688
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Nucleic Acids Research, 2009, Vol. 37, No. 20 6859-6870
© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Time-resolved fluorescence studies of nucleotide flipping by restriction enzymes
1Department of Chemistry, Katholieke Universiteit Leuven, Celestijnenlaan 200F, 3001 Heverlee, Belgium, 2Institute of Biotechnology, Graiciuno 8, LT–02241, Vilnius, Lithuania, 3School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ and 4Collaborative Optical Spectroscopy, Micromanipulation and Imaging Centre (COSMIC), University of Edinburgh, West Mains Road, Edinburgh, EH9 3JZ, UK
*To whom correspondence should be addressed. Tel: +32 16 327399; Fax: +32 16 327990; Email: robert.neely{at}chem.kuleuven.be Correspondence may also be addressed to Anita Jones. Tel. +44 131 6506449; Fax: +44 131 6506453; Email: a.c.jones{at}ed.ac.uk
Received July 2, 2009. Revised August 4, 2009. Accepted August 5, 2009.
Restriction enzymes Ecl18kI, PspGI and EcoRII-C, specific for interrupted 5-bp target sequences, flip the central base pair of these sequences into their protein pockets to facilitate sequence recognition and adjust the DNA cleavage pattern. We have used time-resolved fluorescence spectroscopy of 2-aminopurine-labelled DNA in complex with each of these enzymes in solution to explore the nucleotide flipping mechanism and to obtain a detailed picture of the molecular environment of the extrahelical bases. We also report the first study of the 7-bp cutter, PfoI, whose recognition sequence (T/CCNGGA) overlaps with that of the Ecl18kI-type enzymes, and for which the crystal structure is unknown. The time-resolved fluorescence experiments reveal that PfoI also uses base flipping as part of its DNA recognition mechanism and that the extrahelical bases are captured by PfoI in binding pockets whose structures are quite different to those of the structurally characterized enzymes Ecl18kI, PspGI and EcoRII-C. The fluorescence decay parameters of all the enzyme-DNA complexes are interpreted to provide insight into the mechanisms used by these four restriction enzymes to flip and recognize bases and the relationship between nucleotide flipping and DNA cleavage.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors