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Highly efficient incorporation of the fluorescent nucleotide analogs tC and tC(O) by Klenow fragment

Journal article
Authors Peter Sandin
G. Stengel
Thomas Ljungdahl
Karl Börjesson
Bertil Macao
Marcus Wilhelmsson
Published in Nucleic Acids Research
Volume 37
Issue 12
Pages 3924-3933
ISSN 0305-1048
Publication year 2009
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Pages 3924-3933
Language en
Links dx.doi.org/10.1093/nar/gkp266
https://gup.ub.gu.se/file/71191
Subject categories Biophysical chemistry, Spectroscopy, Biochemistry, Molecular biology

Abstract

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 (tC(O)), 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.

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