Stoichiometry and architecture of active DNA replication machinery in Escherichia coli

Abstract

The multiprotein replisome complex that replicates DNA has been extensively characterized in vitro, but its composition and architecture in vivo is unknown. Using millisecond single-molecule fluorescence microscopy in living cells expressing fluorescent derivatives of replisome components, we have examined replisome stoichiometry and architecture. Active Escherichia coli replisomes contain three molecules of the replicative polymerase, rather than the historically accepted two. These are associated with three molecules of tau, a clamp loader component that trimerizes polymerase. Only two of the three sliding clamps are always associated with the core replisome. Single-strand binding protein has a broader spatial distribution than the core components, with 5 to 11 tetramers per replisome. This in vivo technique could provide single-molecule insight into other molecular machines.

Fig. 1

Slimfield microscopy and photobleach analysis. (A) Slimfield schematic; a laser under-fills the back aperture of an objective lens generating an intense Gaussian field at the sample large enough to image single E. coli. (B) Overlaid brightfield (gray) and 90 ms frame-averaged fluorescence images (yellow) of ε-YPet strain, arrows indicating spots with a stoichiometry of ~3 (cyan) and (C) ~6 (red) ε-YPet molecules, with corresponding single 3 ms frame taken after 45 ms showing that stochastic photobleaching generates different brightnesses. (D) Raw intensity (blue) and filtered (red) for a putative single (left panel) and double (right panel) replisome spot, 45 ms point indicated (arrows), with surface-immobilized YPet in vitro. (E) Fourier spectral analysis for a photobleach trace of the ε-YPet strain with mean±SD peak indicated for brightness of a single YPet.

Fig. 2

Stoichiometries of replisome components and spatial distributions. Stoichiometry distributions per spot (upper panels) using unbiased kernel density estimation for different E. coli strains, N=27-51 cells used in each dataset. Insets indicate examples of overlaid brightfield (gray) and single 3ms fluorescence images (yellow) for each, arrows indicating foci in cells containing two (cyan) and one (red) replisome. 2-Gaussian fits (black) with contributing single Gaussian curves (red and blue) and mean±SD of Gaussian peaks indicated. Lower panels indicate false-color contour plots for 2D averaged spatial distributions for each strain, N=42-151 spots in each dataset. Estimates for mean FWHM <σ> of a symmetrical 2D Gaussian fit and the ratio σ_x/σ_y of the FWHM for the 1D Gaussian fits through the mean spot parallel to the x and y axes are indicated, SD errors in brackets.

Fig. 3

Schematic model for replisome components. (A) Two engaged polymerases, and one of the three β clamps at a distance from the core replisome (circle of diameter 50 nm indicated in gray). The data indicate that ~75% of replisomes have this organization, while ~25% have all three β clamps associated with the core replisome and potentially associated with active PolIII. (B) Expanded view of clamp loader (τ₃δδ′ψχ) and three additional molecules of χψ interacting with Ssb tails. The χψ heterodimer bound to the clamp loader may also contact Ssb (14). γ (shown as a trimer, but the stoichiometry is unknown) then interacts with Ssb-associated χψ.