Single-molecule FRET and linear dichroism studies of DNA breathing and helicase binding at replication fork junctions

Abstract

DNA "breathing" is a thermally driven process in which base-paired DNA sequences transiently adopt local conformations that depart from their most stable structures. Polymerases and other proteins of genome expression require access to single-stranded DNA coding templates located in the double-stranded DNA "interior," and it is likely that fluctuations of the sugar-phosphate backbones of dsDNA that result in mechanistically useful local base pair opening reactions can be exploited by such DNA regulatory proteins. Such motions are difficult to observe in bulk measurements, both because they are infrequent and because they often occur on microsecond time scales that are not easy to access experimentally. We report single-molecule fluorescence experiments with polarized light, in which tens-of-microseconds rotational motions of internally labeled iCy3/iCy5 donor-acceptor Förster resonance energy transfer fluorophore pairs that have been rigidly inserted into the backbones of replication fork constructs are simultaneously detected using single-molecule Förster resonance energy transfer and single-molecule fluorescence-detected linear dichroism signals. Our results reveal significant local motions in the ∼100-μs range, a reasonable time scale for DNA breathing fluctuations of potential relevance for DNA-protein interactions. Moreover, we show that both the magnitudes and the relaxation times of these backbone breathing fluctuations are significantly perturbed by interactions of the fork construct with a nonprocessive, weakly binding bacteriophage T4-coded helicase hexamer initiation complex, suggesting that these motions may play a fundamental role in the initial binding, assembly, and function of the processive helicase-primase (primosome) component of the bacteriophage T4-coded DNA replication complex.

Keywords: DNA helicase; T4 primosome helicase; single-molecule linear dichroism; smFRET; thermal fluctuations.

Fig. 1.

(A) The isolated hexameric gp41 helicase (blue spheres), the subunits of which are bound together by intersubunit (and in this case nonhydrolyzable) NTP ligands (not shown for clarity), binds weakly to the ss–dsDNA junction of a replication fork, favoring dissociation over binding, as shown in B. Introduction of gp61 primase (orange ellipsoid), in a 6:1 gp41-to-gp61 subunit ratio, causes the resulting T4 primosome helicase complex to bind strongly to the replication fork, as shown in C. (D) A hypothetical FES describing local dsDNA fluctuations near the fork junction in the absence of helicase.

Fig. 2.

The smFRET and smFLD experimental layout. (A) The T4 gp41 helicase binds to the d(T)₂₉ loading sequence on the lagging strand of a model DNA replication fork construct. An assembled (gp41)₆∙gp61∙DNA primosome complex can unwind the duplex region of the DNA in the presence of GTP. The strands within the dsDNA region are internally labeled with the FRET donor–acceptor chromophores iCy3 and iCy5, respectively. (B) TIRF excitation scheme and detection method. The polarization of the excitation beam is modulated at 1 MHz. The p-polarization component points in the direction of the y axis, and the s-polarization component is contained within the x–z plane. (C) Orthogonally polarized directions used to measure the FLD signal, from the perspective of the incident beam.

Fig. 3.

(A) Single-molecule iCy3/iCy5 signals were recorded during a T4 primosome–helicase unwinding experiment. An unwinding event occurred at the 17.5-s tick mark. (B) The simultaneously recorded smFLD_r signal exhibits a change in behavior coincident with the unwinding event defined by the smFRET conversion efficiency shown in C. Horizontal dashed lines (red/green) indicate the magnitude of the fluctuations immediately before and after the unwinding event. (Right) Laser polarization ϕ-dependent signal distributions that were used to determine the FLD_r signal shown in B. The donor–acceptor chromophore labels are placed deep within the double-stranded region of the DNA replication fork construct (SI Text, Sample Preparation and Model DNA Replication Fork Constructs).

Fig. 4.

Examples of single-molecule FRET and FLD_r TCFs. TCFs determined from (A) FRET and (B) FLD_r trajectories obtained from a single DNA model fork construct in the absence of helicase proteins. Red lines represent optimized fits to Eq. 4, where the fit parameter is the mean square magnitude of the signal fluctuations, is square average signal, and is the correlation time. The donor–acceptor chromophore labels are placed at the ss–ds fork junction of the DNA construct (SI Text, Sample Preparation and Model DNA Replication Fork Constructs).

Fig. 5.

Dynamics of breathing fluctuations at the replication fork in the absence and presence of helicase. (A) Histograms of relaxation times () obtained from the analysis of smFRET/smFLD trajectories for DNA fork constructs, which were labeled with iCy3/iCy5 placed deep in a duplex region (blue) or at the replication fork junction (red) in the absence of helicase protein. (B) A comparison is shown for a fork-labeled construct in the absence (red) and the presence (blue) of the “frozen” hexameric helicase (gp41 ∙ GTPγS)₆ composed of 300 nM gp41 and 6 μM GTPγS. (C) A comparison is shown of histograms of the relative magnitudes of the fluctuating smFLD_r signal for duplex-labeled DNA (Left), fork-labeled DNA (Center), and fork-labeled DNA + (gp41 ∙ GTPγS)₆ (Right). Histograms of the relaxation times were characterized using the gamma distribution function, given in the text, with skewedness and width parameters α (dimensionless) and β (in units of milliseconds), respectively.