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.

Polymerases:

Topics:

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

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Klenow fragment Sandin P2009 Methods Featured Fluorescence Spectroscopy
Klenow fragment Sandin P2009 Incorporation of non-standard nucleotides 95% + Nucleotide analog: Fluorescent dNTPs
Klenow fragment Sandin P2009 Vmax 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 Sandin P2009 Vmax 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 Sandin P2009 Vmax 8.4 /minute Reaction: Nucleotide incorporation; Substrate: dCTP; Technique: Steady State ; Experimental conditions: pH (pH 8.0), Mg++ (10mM), Temp (37°C)

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.