Nucleic Acids Research Advance Access originally published online on July 7, 2007
Nucleic Acids Research 2007 35(14):4792-4799; doi:10.1093/nar/gkm513
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Nucleic Acids Research, 2007, Vol. 35, No. 14 4792-4799
© 2007 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 |
Nucleotide flipping by restriction enzymes analyzed by 2-aminopurine steady-state fluorescence
1Institute of Biotechnology, Graiciuno 8, LT-02241, Vilnius, Lithuania, 2International Institute of Molecular and Cell Biology, ul. Trojdena 4, 02-109 Warsaw, Poland and 3Max-Planck-Institute for Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01309 Dresden, Germany
*To whom correspondence should be addressed. Tel: +370 5 2602108; Fax: +370 5 2602116; Email: siksnys{at}ibt.lt
Received May 21, 2007. Revised June 14, 2007. Accepted June 14, 2007.
Many DNA modification and repair enzymes require access to DNA bases and therefore flip nucleotides. Restriction endonucleases (REases) hydrolyze the phosphodiester backbone within or in the vicinity of the target recognition site and do not require base extrusion for the sequence readout and catalysis. Therefore, the observation of extrahelical nucleotides in a co-crystal of REase Ecl18kI with the cognate sequence, CCNGG, was unexpected. It turned out that Ecl18kI reads directly only the CCGG sequence and skips the unspecified N nucleotides, flipping them out from the helix. Sequence and structure conservation predict nucleotide flipping also for the complexes of PspGI and EcoRII with their target DNAs (/CCWGG), but data in solution are limited and indirect. Here, we demonstrate that Ecl18kI, the C-terminal domain of EcoRII (EcoRII-C) and PspGI enhance the fluorescence of 2-aminopurines (2-AP) placed at the centers of their recognition sequences. The fluorescence increase is largest for PspGI, intermediate for EcoRII-C and smallest for Ecl18kI, probably reflecting the differences in the hydrophobicity of the binding pockets within the protein. Omitting divalent metal cations and mutation of the binding pocket tryptophan to alanine strongly increase the 2-AP signal in the Ecl18kI–DNA complex. Together, our data provide the first direct evidence that Ecl18kI, EcoRII-C and PspGI flip nucleotides in solution.
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