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.

Figure 1.

Chemical structure and base pairing pattern of the fluorescent base analogs tC and tC^O. Absorption and emission spectra of the free monomers of tC (dashed line) and tC^O (solid line). The fluorescence emission is normalized to represent the relative quantum yields.

Figure 2.

Incorporation of fluorescent nucleotide analogs by Klenow fragment. (a) Incorporation of dCTP (top), dtCTP (middle) and dtC^OTP (bottom) into the depicted primer/template with the templating base X being G, T, C or A. For primer extension, 16.7 μM nucleotide was reacted with 2 μM primer/template in the presence of 11.6 nM Klenow fragment. The reaction was stopped after 2 min by addition of formaldehyde and 50 mM EDTA. Control reactions without nucleotides are shown in the left lane. (b) Read-through assays of triphosphates dCTP (first lane), dtCTP (second lane) and dtC^OTP (third lane). The assay conditions were identical to (a) except that 16.7 μM of each nucleotide was added. Primer extension in the presence of dATP only (fourth lane). DNA strands were visualized by staining with SYBRgold.

Figure 3.

Competitive elongation experiments. Primer extension was performed in the presence of dCTP and dtCTP (top, left column) or dCTP and dtC^OTP (bottom left column) at different mixing ratios. The concentration of dCTP is given below the gels and the corresponding nucleotide analog concentration is 100% – (% dCTP). The products were analyzed by gel electrophoresis and the product bands stained with SYBRgold. The reaction conditions were like in Figure 2 with a total nucleotide concentration of 83.4 μM. The panel on the right displays the percentage of primer that was elongated by dtCTP (top) or by dtC^OTP (bottom) upon varying the mole fraction of dCTP.

Figure 4.

Formation of the ternary KF–DNA–dNTP complex probed by steady-state fluorescence anisotropy. (a) In three separate experiments, 125 nM (black triangles), 250 nM (gray circles) and 500 nM (black squares) dtCTP was incubated with increasing amounts of KF in the presence of 10 μM primer/template (X = G). In all binding experiments, the 3′-end of the primer was terminated with a 2′,3′-dideoxy-cytosine. (b) The same experiments were carried out using 75 nM (black triangles), 125 nM (gray circles) and 250 nM (black squares) dtC^OTP. The affinity curves resulting from the best fits (see text) are shown as solid curves.

Figure 5.

Competitive binding curves. The samples contained 10 µM primer/template (X = G), 203 nM KF and 250 nM dtCTP (a) or 250 nM dtC^OTP (b). The stepwise addition of dCTP (black squares) replaces the fluorescent nucleoside triphosphates in the ternary complexes. Insets: Effect of protein binding on the fluorescence emission of dtCTP (inset in a) and dtC^OTP (inset in b). Total 125 nM nucleotide analog were incubated with 284 nM Klenow fragment and 10 µM primer/template (X = G) until the fluorescence remained stable. Spectra of the free nucleotide analogs are shown as black lines and spectra of the nucleotides in complex with KF are shown as black dots.