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

Author

Peter Sandin

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Gudrun Stengel

University of Colorado at Boulder

Thomas Ljungdahl

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Karl Börjesson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Bertil Macao

University of Gothenburg

Marcus Wilhelmsson

Chalmers, Chemical and Biological Engineering, Physical Chemistry

Nucleic Acids Research

0305-1048 (ISSN) 1362-4962 (eISSN)

Vol. 37 12 3924-3933

Subject Categories

Physical Chemistry

Biochemistry and Molecular Biology

DOI

10.1093/nar/gkp266

More information

Latest update

3/6/2018 8