Large domain movements upon UvrD dimerization and helicase activation

Abstract

Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.

Keywords: DNA motors; DNA repair; conformational heterogeneity; single molecule.

Fig. 1.

The 2B subdomain of UvrD populates at least four discrete rotational conformational states. (A) The 2B subdomain (blue) in the open and closed states in crystal structures. Movement of the 2B domain is monitored by the change in FRET efficiency between a donor (Cy3) and an acceptor (Cy5) on the 1B (A100C) and the 2B (A473C) subdomains. (B) A single-molecule time trace [Cy3 (green), Cy5 (red), and FRET efficiency (blue)] in imaging buffer plus 60 mM NaCl (25.0 °C) shows anticorrelated changes in Cy3 and Cy5 with a constant total intensity (black). Transitions are observed among four FRET states (S1 to S4) as shown by the hidden Markov fit (solid black line) to the trajectory. (C) Schematic of the four FRET substates and their interconversion rates obtained from hidden Markov analyses.

Fig. 2.

Population distribution of conformational states shifts to the more open states with increasing NaCl concentration. (A) Normalized histograms (imaging buffer) showing FRET efficiencies and species fractions of the 2B substates at [NaCl] of 20 mM (n = 145), 40 mM (n = 102), 60 mM (n = 165), 80 mM (n = 73), 200 mM (n = 94), 400 mM (n = 76), and 600 mM (n = 89). The high FRET states (S3 and S4) are more populated at low [NaCl], and the low FRET states (S1 and S2) are more populated at high [NaCl]. (B) FRET efficiencies of the four substates are independent of [NaCl]. (C) Relative populations of the four FRET substates change with [NaCl]. (D) Average FRET efficiencies from single-molecule experiments (filled circles) overlaid with the normalized FRET efficiencies from ensemble studies (35) show a net change from a closed to an open state with increasing [NaCl] (60 mM NaCl midpoint).

Fig. 3.

Effects of binding of DNA, ATP-ɣ-S, and a second UvrD molecule on the 2B conformational state distribution. (A) FRET histogram of labeled UvrD(A100C, A473C) with 0.5 mM ATP-ɣ-S (n = 72) or without (n = 55). (B) FRET histogram upon addition of 300 nM 3′(dT)₂₀ds18 bp to A with (n = 48) or without ATP-ɣ-S (n = 89). (C) FRET histogram upon addition of 0.5 µM wtUvrD to B greatly enhances the S4 population in the presence (n = 54) or absence (n = 69) of ATP-ɣ-S. (D) FRET histogram upon addition of 0.5 µM UvrD(K35I) to B (n = 43). (E) FRET histogram upon addition of 1.0 µM Rep to B (n = 45). (F) Populations of the four substates for the experiments in A–C with or without ATP-ɣ-S. (G) Populations of the four substates for the experiments in C–E in the presence of ATP-ɣ-S.

Fig. 4.

Unwinding of DNA by a UvrD dimer. (A) Schematic of the expected DNA unwinding time course phases. Phase I is binding of Cy3/Cy5-DNA to UvrD on the surface. Phase II (magenta) is binding of a second UvrD plus ATP results in DNA unwinding with anticorrelated changes in Cy3 and Cy5 resulting in a FRET increase. Phase III (cyan) is a brief plateau in FRET efficiency. Phase IV is release of the Cy5 DNA strand. Experimental DNA unwinding trajectories and histograms of unwinding times at (B) 5 µM (n = 37), (C) 10 µM (n = 35), and (D) 25 µM ATP (n = 29). (E) Histograms were fit to a gamma distribution to obtain the DNA unwinding parameters as a function of [ATP].