Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.

Sandin P, Stengel G, Ljungdahl T, Börjesson K, Macao B, Wilhelmsson LM

Nucleic acids research (2009), Volume 37, Page 3924

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Abstract:

Studies of the mechanisms by which DNA polymerases select the correct ...

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.

Polymerases:

Klenow fragment

Topics:

Kinetic Parameters, Nucleotide Analogs / Template Lesions, Nucleotide Incorporation, Methods

Status:

Results:

Polymerase	Reference	Property	Result	Context
Klenow fragment	Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.	Methods Featured	Fluorescence Spectroscopy
Klenow fragment	Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.	Incorporation of non-standard nucleotides	95% +	Nucleotide analog: Fluorescent dNTPs
Klenow fragment	Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.	V_max	8.4 /minute	Reaction: Nucleotide incorporation; Substrate: CTP analog; Technique: Steady State (dtCoTP fluor analogue); Experimental conditions: pH (pH 8.0), Mg++ (10mM), Temp (37°C)
Klenow fragment	Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.	V_max	5.4 /minute	Reaction: Nucleotide incorporation; Substrate: CTP analog; Technique: Steady State (dtCTP fluor analogue); Experimental conditions: pH (pH 8.0), Mg++ (10mM), Temp (37°C)
Klenow fragment	Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment.	V_max	8.4 /minute	Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State ; Experimental conditions: pH (pH 8.0), Mg++ (10mM), Temp (37°C)