![]() ![]() 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. 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. 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. 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. Although the kinetics of the Klenow fragment of Escherichia coli DNA Polymerase I (Pol I(KF)) have been studied using a variety of approaches (13, 15 20), there are conflicting reports of the synthesis rate and it has not yet been possible to dissect the interplay between pausing and burst synthesis. 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. ![]() The 3'5' exonuclease activity of the enzyme is eliminated by mutations in the 3'5'-exonuclease active site. It exhibits 5'3' polymerase activity, but lacks the 3'5' and 5'3' exonuclease activities of DNA Polymerase I. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA-protein interaction than do tethered fluorophores. Thermo Scientific Klenow Fragment, exo-, is the large fragment of DNA polymerase I. 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. ![]()
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