Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization - PubMed (original) (raw)

Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization

H Q Xu et al. Nucleic Acids Res. 2003.

Abstract

A new method for helicase-catalyzed DNA unwinding is described. This assay takes advantage of the substantial change in fluorescence polarization (FP) upon helicase binding and DNA unwinding. The low anisotropy value, due to the fast tumbling of the free oligonucleotide in solution, increases abruptly upon binding of helicase to the fluorescein-labeled oligonucleotide. The high anisotropy of the helicase- DNA complex decreases as the fluorescein-labeled oligonucleotide is released from the complex through helicase-catalyzed DNA unwinding. This FP signal can be measured in real time by fluorescent spectroscopy. This assay can simultaneously monitor DNA binding and helicase-catalyzed DNA unwinding. It can also be used to determine the polarity in DNA unwinding mediated by helicase. This FP assay should facilitate the study of the mechanism by which helicase unwinds duplex DNA, and also aid in screening for helicase inhibitors, which are of growing interest as potential anticancer agents.

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Figures

Figure 1

Figure 1

(A) Schematic illustration of the FP-based unwinding assay. A fluorescein-labeled oligonucleotide (open circles) was annealed to the proximal end of a ssDNA molecule. This substrate alone gives a low anisotropy value. The anisotropy signal increases upon the binding of helicase (closed circles) to this DNA substrate. When the DNA strands are unwound by a helicase, the fluorescein-labeled oligo is released from the DNA–helicase complexes, and the anisotropy value is even lower than partial duplex DNA substrate. The unwinding reaction is followed by measurement of the anisotropy value. (B) DNA unwinding followed by fluorescence anisotropy. Fluorescein-labeled partial duplex DNA (5 nM) (black closed circles) was incubated in the unwinding buffer. The anisotropy value was increased and then decreased upon addition of 30 nM RecQ and 1 mM ATP, respectively.

Figure 2

Figure 2

Effect of fluorescein-labeled DNA length protein and ATP concentration on the kinetics of duplex unwinding. (A) Kinetics of duplex unwinding with DNA formed with different lengths of fluorescein-labeled ssDNA as indicated. A 5 nM concentration of each DNA substrate and 40 nM RecQ protein were used in this study. (B) DNA substrate E (2 nM) (36 base duplex DNA) was incubated with 50 and 200 nM RecQ helicase. The DNA unwinding reaction was initiated by adding either 1 mM ATP (closed squares and open circles) or 1 mM ATP containing 2 µM ssDNA trap (closed circles). (C) DNA substrate A (2 nM) was incubated with 40 nM RecQ helicase. The unwinding reaction was initiated by the addition of different concentrations of ATP as indicated.

Figure 3

Figure 3

(A) Effects of nucleotides on RecQ helicase-catalyzed DNA unwinding activity. DNA unwinding assays were performed as described in Materials and Methods using DNA substrate A (2 nM); each reaction contained 40 nM helicases and one of the listed nucleotides at a final concentration of 1 mM. The data presented are the average of three independent determinations. (B) Helicase-mediated DNA unwinding activity is ATP dependent. DNA substrate A (5 nM) was incubated with either 30 nM RecQ helicase or 30 nM ATPase-deficient mutant (K55A) enzyme and the unwinding reaction was initiated by addition of either 1 mM ATP (closed circles, wt helicase; open squares, mutant helicase) or 1 mM ATP containing 5 mM EDTA (closed squares).

Figure 4

Figure 4

RecQ helicase resides on the loading strand of DNA whereas the leading strand is excluded from the DNA–helicase complexes during the unwinding reaction. DNA substrates A, C, D (5 nM) were incubated with 30 nM RecQ helicase at 25°C and 1 mM ATP was then added. The anisotropy values were recorded every 8 s.

Figure 5

Figure 5

The effect of duplex DNA length on the single-turnover kinetics of RecQ-mediated DNA unwinding. RecQ helicase (50 nM) was incubated with 2 nM DNA substrtate A (circles, 14 bases in duplex length), substrate B (squares, 22 bases in duplex length), substrate E (rhombus, 40 bases in duplex length); DNA unwinding was initiated upon addition of 1 mM ATP containing 2 µM ssDNA trap at 25°C. Data from all three time courses were fitted to the exponential equation: A t = A exp(–k_obs_t), where A t is the anisotropy amplitude at time t, and _k_obs is the observed rate constant. The insert shows the fraction unwound of DNA substrates.

Figure 6

Figure 6

Arrhenius plots of the observed unwinding rate of RecQ helicase. The experiments were performed under standard conditions at temperatures between 4 and 37°C. DNA substrates A and B (2 nM) and 40 nM RecQ helicase were used.

Figure 7

Figure 7

Helicase polarity determination by fluorescence anisotropy assay. RecQ helicase (30 nM) or UvrD helicase (30 nM) was incubated, respectively, with 2 nM partial duplex DNA substrate F or G, and DNA unwinding was initiated by the addition of 1 mM ATP.

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