Dynamics of site switching in DNA polymerase

Abstract

DNA polymerases replicate DNA by catalyzing the template-directed polymerization of deoxynucleoside triphosphate (dNTP) substrates onto the 3' end of a growing DNA primer strand. Many DNA polymerases also possess a separate 3'-5' exonuclease activity that is used to remove misincorporated nucleotides from the nascent DNA (proofreading). The polymerase (pol) and exonuclease (exo) activities are spatially separated in different enzyme domains, indicating that a mechanism must exist to transfer the growing primer terminus from one site to the other. Here we report a single-molecule Förster resonance energy transfer (smFRET) system that directly monitors the movement of a DNA substrate between the pol and exo sites of DNA polymerase I Klenow fragment (KF). FRET trajectories recorded during the encounter between single polymerase and DNA molecules reveal that DNA can channel between the pol and exo sites in both directions while remaining closely associated with the enzyme (intramolecular transfer). In addition, it is evident from the trajectories that DNA can also dissociate from one site and subsequently rebind at the other (intermolecular transfer). Rate constants for each pathway have been determined by dwell-time analysis, revealing that intramolecular transfer is the faster of the two pathways. Unexpectedly, a mispaired primer terminus accesses the exo site more frequently when dNTP substrates are also present in solution, which is expected to enhance proofreading. Together, these results explain how the separate pol and exo activities of KF are physically coordinated to achieve efficient proofreading.

Figure 1

Cocrystal structures of DNA polymerase bound to DNA. The structure on the left corresponds to the open binary complex of the KF homolog Bst Pol I and DNA (PDB code 1L3S). The primer 3′ terminus is located in the pol site (P). The structure on the right is for a complex of KF with DNA bound at the exo site (E) (PDB code 1KLN). In contrast to the structure shown on the left, the DNA contains a single-stranded extension on the primer strand rather than the template strand. In both structures, the primer strand is yellow, the template strand is orange, the polymerase domain is grey and the 3′–5′ exonuclease domain is aqua. The donor and acceptor probes used for smFRET measurements are shown as green and red spheres, respectively. The mechanism for transfer of DNA from the pol site to the exo site is not fully understood and is the subject of the present study.

Figure 2

Binding, dissociation and site switching of KF visualized by smFRET. The DNA contains a G•G mismatch at the primer 3′ terminus (1 mm, Table 1). (a) Time traces of the A488 donor (green) and A594 acceptor (red) emission intensity, recorded with 100 ms integration time. The corresponding FRET efficiency trajectory (blue) is shown in the lower panel. The dashed lines at 0, 0.65 and 0.80 FRET efficiencies correspond to unbound DNA, DNA bound at the exo site and DNA bound at the pol site, respectively. (b) FRET trajectory for the same sample recorded using 33 ms integration time, showing multiple pol-exo switching events. (c) Histogram of FRET efficiencies for wt KF (upper panel, compiled from 230 trajectories) and L361A KF (lower panel, compiled from 261 trajectories), each interacting with the mispaired primer/template. In the upper panel, the dashed black lines are Gaussian fits to each peak and the red line is the composite fit to the overall histogram. The fitted centers and widths are 0.67 and 0.09, respectively, for the lower FRET peak, and 0.80 and 0.08, respectively, for the higher FRET peak. The fractional areas enclosed by each peak are indicated. In the lower panel, the black line is the best fit to a single Gaussian function, with fitted center and width of 0.79 and 0.10, respectively. (d) Transition probability density plot for wt KF interacting with the mispaired primer/template. The colored regions reveal connectivity between individual FRET states, with shading from blue to red to yellow indicating higher transition probability.

Figure 3

Kinetics of intramolecular switching of DNA between the pol and exo sites of KF. (a) Histogram of dwell-times in the pol site before transition to the exo site for wt KF interacting with primer/template containing a terminal G•G mispair (1 mm, Table 1). The histogram is compiled from 1195 transitions. The solid line is the best fit to a single exponential function, with the indicated rate constant. (b) Corresponding dwell-time histogram (compiled from 1584 transitions) for wt KF interacting with the mispaired primer/template in the presence of 1 mM dTTP. The exponential fit line and corresponding rate constant are indicated. (c) Corresponding dwell-time histogram (compiled from 939 transitions) for wt KF interacting with the mispaired primer/template in the presence of 1 mM dATP. The exponential fit line and corresponding rate constant are indicated. (d) Representative smFRET trajectory for wt KF interacting with the mispaired primer/template in the presence of dTTP (1 mM). (e) Representative smFRET trajectory for wt KF interacting with fully base paired primer/template (0 mm, Table 1) in the presence of dTTP (1 mM). In (d) and (e), the dashed lines at 0, 0.65 and 0.80 FRET efficiencies correspond to unbound DNA, DNA bound at the exo site and DNA bound at the pol site, respectively.

Figure 4

Two pathways for switching of DNA between the pol and exo sites of KF as revealed by the present smFRET studies. The rate constants for each step in the two pathways are indicated. The orange circle at the end of the DNA primer/template represents the biotin group used for surface immobilization.