title: Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.
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.
authors.affiliation: Department of Chemical and Biological Engineering/Physical Chemistry, Chalmers University of Technology, S-41296 Gothenburg, Sweden.
authors.forename: Peter
authors.initials: P
authors.lastname: Sandin
authors.forename: Gudrun
authors.initials: G
authors.lastname: Stengel
authors.forename: Thomas
authors.initials: T
authors.lastname: Ljungdahl
authors.forename: Karl
authors.initials: K
authors.lastname: Börjesson
authors.forename: Bertil
authors.initials: B
authors.lastname: Macao
authors.forename: L Marcus
authors.initials: LM
authors.lastname: Wilhelmsson
mesh_terms: D001667:Binding, Competitive; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry; D004256:DNA Polymerase I / Q000378:metabolism*; D017931:DNA Primers; D003853:Deoxyribonucleosides / Q000138:chemical synthesis / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D010078:Oxazines / Q000737:chemistry* / Q000378:metabolism; D010640:Phenothiazines / Q000737:chemistry* / Q000378:metabolism
chemical_list: C492077:1,3-diaza-2-oxophenothiazin-3-ylacetic acid; C526502:1,3-diaza-2-oxophenoxazine; D017931:DNA Primers; D003853:Deoxyribonucleosides; D005456:Fluorescent Dyes; D010078:Oxazines; D010640:Phenothiazines; D004247:DNA; D004256:DNA Polymerase I