Breathing fluctuations in position-specific DNA base pairs are involved in regulating helicase movement into the replication fork

Abstract

We previously used changes in the near-UV circular dichroism and fluorescence spectra of DNA base analogue probes placed site specifically to show that the first three base pairs at the fork junction in model replication fork constructs are significantly opened by "breathing" fluctuations under physiological conditions. Here, we use these probes to provide mechanistic snapshots of the initial interactions of the DNA fork with a tight-binding replication helicase in solution. The primosome helicase of bacteriophage T4 was assembled from six (gp41) helicase subunits, one (gp61) primase subunit, and nonhydrolyzable GTPγS. When bound to a DNA replication fork construct this complex advances one base pair into the duplex portion of the fork and forms a stably bound helicase "initiation complex." Replacement of GTPγS with GTP permits the completion of the helicase-driven unwinding process. Our spectroscopic probes show that the primosome in this stable helicase initiation complex binds the DNA of the fork primarily via backbone contacts and holds the first complementary base pair of the fork in an open conformation, whereas the second, third, and fourth base pairs of the duplex show essentially the breathing behavior that previously characterized the first three base pairs of the free fork. These spectral changes, together with dynamic fluorescence quenching results, are consistent with a primosome-binding model in which the lagging DNA strand passes through the central hole of the hexagonal helicase, the leading strand binds to the "outside" surfaces of subunits of the helicase hexamer, and the single primase subunit interacts with both strands.

Fig. 1.

Spectroscopic properties of DNA constructs as a function of 2-AP probe position relative to the ss–dsDNA junction. Fluorescence intensities at 370 nm for constructs with single 2-AP probes in the (A) lagging and (B) leading DNA strands. All constructs are shown in Table S1 and identified by construct name below each fluorescence intensity bar in Fig. 1 A–D and by color in the spectra of Fig. 1 E and F. Fluorescence intensities at 370 nm are shown for constructs with 2-AP dimer probes in the (C) lagging and (D) leading DNA strands. Fluorescence intensity changes have been normalized to the intensity of the probe signal in the control ssDNA, and full fluorescence spectra are shown in Fig. S2. CD spectra of forked DNA constructs with 2-AP dimer probes at defined positions on the (E) lagging and (F) leading strands.

Fig. 2.

Fluorescence intensity changes caused by helicase binding for forked DNA constructs with 2-AP monomer probes in the lagging (5^′ → 3^′) strand. (A) Intensities at 370 nm for ssDNA and for constructs with 2-AP monomer probes located directly at the ss–dsDNA fork junction. (B) Intensities for 2-AP monomer probes located deeper within the dsDNA portion of the construct. The first bar (red) in each panel corresponds to the fluorescence intensity of the free construct, the second (blue) shows the fluorescence intensity of that construct in the presence of the hexameric gp41–GTP γS complex, the third (green) shows the intensity in the presence of the primosome helicase, and the fourth (black, when present) corresponds to the intensity in the presence of a gp41 helicase hexamer in the presence of excess GTP. The designation under each group of fluorescent intensities along the x axis identifies the construct and probe position(s) used.

Fig. 3.

Fluorescence intensity changes caused by helicase binding for forked DNA constructs with 2-AP monomer probes in the leading (3^′ → 5^′) strand. (A) Intensities at 370 nm for ssDNA and for constructs with 2-AP monomer probes located directly at the ss–dsDNA fork junction. (B) Intensities for 2-AP monomer probes located deeper within the dsDNA portion of the construct. Color coding is the same as in Fig. 2.

Fig. 4.

Acrylamide quenching of 2-AP monomer probes in the leading and lagging strands of DNA fork constructs. (A) Forked DNA construct with the 2-AP monomer probe at position -1 on the lagging strand (free DNA, filled circles; helicase-bound DNA, open circles) and at position 1 (free DNA, filled squares; helicase-bound DNA, open squares). (B) Forked DNA constructs with the 2-AP monomer probe at position -1 on the leading strand (free DNA, filled circles; helicase-bound DNA, open circles) and at position 1 (free DNA, filled squares; helicase-bound DNA, open squares). (C) The ssDNA strands with the 2-AP monomer probe on the 5′ strand (free DNA, filled circles; helicase-bound DNA, open circles) and on the 3′ strand (free DNA, filled squares; helicase-bound DNA, open squares). Closed symbols correspond to free DNA alone and open symbols to helicase–DNA complexes. The arrows point from the free to the helicase-bound lines of the Stern–Vollmer plots for the same DNA construct in each case.

Fig. 5.

Proposed unwinding mechanism of the T4 primosome helicase. The constituents of the primosome are gp41 subunits (blue ellipses), gp61 subunit (red ellipses), GTP (yellow rectangles), and GDP (red rectangles). The “degree of openness” of the base pairs adjacent to the ss–dsDNA junction is indicated in each panel, and the numbers below each DNA construct represent the numbering of the various base pairs prior to initial helicase binding. Step A: The GTP-bound gp41 hexameric helicase loads onto the free DNA fork construct and the gp61 primase subunit binds and stabilizes the complex at the fork junction. Positioning is facilitated by the uniquely unstacked conformation of the -1 bases. As a result of this initial binding the first duplex (breathing) base pair at position 1 is fully unwound and the breathing of the base pairs at initial positions 2, 3, and 4 are all enhanced. Step B: GTP hydrolysis occurs at the gp41–gp41 interface positioned adjacent to the bound gp61 subunit, destabilizing that subunit interface and permitting the primosome to “capture” the now unwound first base pair of the original duplex. This base pair becomes the new -1 position, thereby moving the breathing properties of each base pair at the fork one position further into the duplex sequence. The gp41 hexamer rotates by one subunit (approximately 60°) and the primase translocates to the next gp41–gp41 interface. Step C: The GDP (and P_i) hydrolysis products formed in step B dissociate, a new GTP binds and stabilizes the previously destabilized gp41–gp41 subunit interface, and the primosome helicase is ready to begin a new unwinding-rotation-hydrolysis cycle.