Nucleic Acids Research Advance Access originally published online on April 28, 2009
Nucleic Acids Research 2009 37(12):3924-3933; doi:10.1093/nar/gkp266
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Nucleic Acids Research, 2009, Vol. 37, No. 12 3924-3933
© 2009 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.
Molecular Biology |
Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment
1Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, S-41296 Gothenburg, Sweden, 2Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, USA and 3Department of Medical Biochemistry, University of Gothenburg, PO Box 440, S-405 30 Gothenburg, Sweden
*To whom correspondence should be addressed. Tel: +46 31 7723051; Fax: +46 31 7723858; Email: marcus.wilhelmsson{at}chalmers.se
Correspondence may also be addressed to Gudrun Stengel. Tel: +1 303 4923591; Fax: +1 303 4925894; Email: gudrun.stengel{at}colorado.edu
Received March 6, 2009. Revised April 9, 2009. Accepted April 9, 2009.
Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5'-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.