Symmetric activity of DNA polymerases at and recruitment of exonuclease ExoR and of PolA to the Bacillus subtilis replication forks

Abstract

DNA replication forks are intrinsically asymmetric and may arrest during the cell cycle upon encountering modifications in the DNA. We have studied real time dynamics of three DNA polymerases and an exonuclease at a single molecule level in the bacterium Bacillus subtilis. PolC and DnaE work in a symmetric manner and show similar dwell times. After addition of DNA damage, their static fractions and dwell times decreased, in agreement with increased re-establishment of replication forks. Only a minor fraction of replication forks showed a loss of active polymerases, indicating relatively robust activity during DNA repair. Conversely, PolA, homolog of polymerase I and exonuclease ExoR were rarely present at forks during unperturbed replication but were recruited to replications forks after induction of DNA damage. Protein dynamics of PolA or ExoR were altered in the absence of each other during exponential growth and during DNA repair, indicating overlapping functions. Purified ExoR displayed exonuclease activity and preferentially bound to DNA having 5' overhangs in vitro. Our analyses support the idea that two replicative DNA polymerases work together at the lagging strand whilst only PolC acts at the leading strand, and that PolA and ExoR perform inducible functions at replication forks during DNA repair.

Figure 1.

Localization of polymerases during exponential growth. Subcellular localization by epifluorescence in representative untreated live Bacillus subtilis cells. The overlay panels show DnaX-CFP in red and PolC-mV, DnaE-mV, PolA-mV and ExoR-mV in green. Scale bar: 2 μm.

Figure 2.

Altered localization patterns after induction of DNA damage. Localization by epifluorescence in representative live Bacillus subtilis cells after induction of DNA damage with MMC. The overlay panels show DnaX-CFP in red and PolC-mV, DnaE-mV, PolA-mV, and ExoR-mV in green. Note that DnaX-CFP foci rarely colocalize with PolA-mV or ExoR-mV foci. Scale bar: 2 μm.

Figure 3.

Diffusion patterns of DNA polymerases. GMM analyses of frame-to-frame displacements in x- and y-directions. (A) PolC-mV (B) DnaE-mV. Red lines represent the sum of the two Gaussian distributions. Dotted and dashed lines represent the single Gaussian distributions corresponding to the static and mobile fractions. Bubble plots show a comparison of fraction sizes (size of the bubble) and diffusion constants (y-axis), between different growth conditions: distribution in untreated cells (dark blue circles), in MMC-treated (yellow) and in UV-treated (light blue circles) cells. Step size distributions reveal two populations for each protein, a mobile (upper circles) and a static (lower circles) fraction.

Figure 4.

Diffusion patterns of PolA and of ExoR. GMM analyses of frame-to-frame displacements in x- and y-directions. (A) PolA-mV, (B) ExoR-mV, (C) PolA-mV ΔexoR (D) ExoR-mV ΔpolA. See Figure 3 for explanations.

Figure 5.

Scatter plot and significance test results from dynamics and fraction sizes of polymerases. Scatter plot showing the relationship between the diffusion coefficients (D_i) of PolA-mV, ExoR-mV, ΔpolA ExoR-mV and ΔexoR PolA-mV (y-axis) and comparison of fraction sizes (x-axis), of (A) fast and (B) slow-diffusing populations. (C) Results of the hypothesis testing to the differences in the fraction sizes of all proteins in terms of P-value: As usual, *, ** and *** stand for P-values lower than 0.1, 0.05 and 0.01, respectively, whilst n.s. stands for statistically not significant. The most relevant comparisons are highlighted in red.

Figure 6.

Diffusion patterns of DnaX-mV compared to PolC-mV and DnaE-mV. (A) GMM analyses of frame-to-frame displacements in x- and y-directions of DnaX-mV, PolC-mV and DnaE-mV. Black lines represent the sum of the two Gaussian distributions. Dotted red and blue lines represent the single Gaussian distributions corresponding to the static and mobile fractions. (B) Bar plot with error bars shown illustrates fractions sizes in untreated cells and their error according to the 95% confidence intervals of the fit. Inside in white, each Diffusion coefficient in μm·s⁻². As usual, *, ** and *** stands for p-values lower than 0.1, 0.05 and 0.01 respectively, whilst n.s. stands for statistically not significant according to a two-sample Kolmogorov–Smirnov significance test on the step size distributions. Note that the same diffusion constants for DnaE and PolC were chosen (these do not differ markedly from the actual diffusion constants), which slightly adapts the sizes of static and dynamic fractions, but allows for a direct comparison between the fraction sizes of the two proteins.

Figure 7.

Dwell times. (A) Relative differences of each strain once applied MMC and UV treatment respect to no treatment. (B) Average residence times (± standard error of the mean) of PolC-mV, DnaE-mV, PolA-mV, ExoR-mV, ExoR-mV ΔpolA and PolA-mV ΔexoR strains, before and after treatment with MMC or UV. Dwell times are estimated using an exponential decay model. Histograms show events of resting fitted by a two-component exponential function. *, ** and *** stand for P-values lower than 0.1, 0.05 and 0.01, respectively, whilst n.s. stands for statistically not significant changes on the dwell times distributions.

Figure 8.

Localization of PolA and ExoR relative to replication forks. A) Location of DnaX-CFP and overlaid tracks from PolA-mV or from ExoR-mV in representative Bacillus subtilis cells untreated and after induction of DNA damage with MMC or UV light as indicated. (B) Distance of PolA-mV or ExoR-mV tracks relative to a DnaX-CFP focus, which is positioned at ‘0 μm’. Panels correspond to those in (A), such that the position of a second or third DnaX-CFP focus can be inferred after induction of DNA damage by the additional accumulation of static tracks far from the ‘0’ position. (C) Histogram showing the distances of PolA-mV and ExoR-mV trajectories to the location of DnaX-CFP in 50 cells, treated or untreated.

Figure 9.

DNA binding of ExoR. EMSAs showing ExoR binding to different nucleotide substrates. ExoR binding specifically to dsDNA panels A, D and E or ssDNA (panel B), or to RNA (panel C and F). EMSAs were performed with increasing amounts (0–750 nM) of purified ExoR and fragments of 68 bp, containing either DNA or RNA, generated by annealing of custom-made oligonucleotides. Samples were mixed with loading buffer and analysed through 6% (v/v) native polyacrylamide gels. Lanes labelled ‘0’ show the control substrate in the absence of protein. ExoR shows highest binding affinity to 5′ overhangs in dsDNA (panel A). Lines below each panel represent DNA in blue or RNA in red. (G) Plot of the measurement of the relative intensities of the two bands in the EMSA gels for each DNA/RNA substrate.