Replisome activity slowdown after exposure to ultraviolet light in Escherichia coli

Abstract

The replisome is a multiprotein machine that is responsible for replicating DNA. During active DNA synthesis, the replisome tightly associates with DNA. In contrast, after DNA damage, the replisome may disassemble, exposing DNA to breaks and threatening cell survival. Using live cell imaging, we studied the effect of UV light on the replisome of Escherichia coli Surprisingly, our results showed an increase in Pol III holoenzyme (Pol III HE) foci post-UV that do not colocalize with the DnaB helicase. Formation of these foci is independent of active replication forks and dependent on the presence of the χ subunit of the clamp loader, suggesting recruitment of Pol III HE at sites of DNA repair. Our results also showed a decrease of DnaB helicase foci per cell after UV, consistent with the disassembly of a fraction of the replisomes. By labeling newly synthesized DNA, we demonstrated that a drop in the rate of synthesis is not explained by replisome disassembly alone. Instead, we show that most replisomes continue synthesizing DNA at a slower rate after UV. We propose that the slowdown in replisome activity is a strategy to prevent clashes with engaged DNA repair proteins and preserve the integrity of the replication fork.

Keywords: DNA replication; Escherichia coli; UV; fluorescence microscopy; replisome.

Fig. 1.

Unbinding of replisome subunits at a UV lesion. (A) Model of the architecture of an active replisome. DNA is unwound by the DnaB helicase. DnaG primase binds to DnaB. Pol III and the clamp loader bind each other through the τ subunit of the clamp loader, which also mediates binding to the DnaB helicase. The β-clamp is left behind the fork after the completion of an Okazaki fragment at the lagging strand. SSB covers ssDNA produced during the cycle of lagging strand synthesis. (B) Representative images of cells carrying YPet-DnaB or ε-YPet before, and 5 min after, exposure to 25 J/m² UV dose. White arrows mark the location of foci. (Scale bar: 1 μm.) (C) Average number of the foci per cell of YPet-DnaB or ε-YPet 5 min after exposure to different UV doses. (D) Distribution of the number of foci per cell in cells carrying YPet-DnaB or ε-YPet 5 min after exposure to UV. (E) Average number of foci for all of the replisome subunits tested: YPet-DnaB, YPet-DnaG, YPet-β, ε-YPet, τ-YPet, χ-YPet, and SSB-YPet. Pictures were taken 5 min after exposure. Error bars represent SE.

Fig. 2.

DnaB remains in proximity to Pol III after UV. (A) Representative images of a strain carrying YPet-DnaB and ε-mTagRFP before, and at various times after, UV treatment. White arrows mark colocalization of foci in the two channels. (Scale bar: 1 μm.) (B) Distribution of apparent distances between a YPet-DnaB focus and the closest ε-mTagRFP focus in a cell. The untreated sample is compared with the results at 10, 30, and 60 min after UV. The median (M) of each population is shown. (C) Average number of foci per cell for YPet-DnaB and ε-mTagRFP before (0 min) and at various times after treatment. (D) Number of cells with at least one fluorescent focus for YPet-DnaB or ε-mTagRFP at various times after treatment. (E) Estimation of helicase disassembly at various times after UV treatment. (Inset) Numbers are based on the difference between a simulated growth of foci (green line), assuming no dissasembly, and the data of YPet-DnaB from Fig. 1C and C (yellow line). (F) Number of PriA-mNeonGreen (green) and YPet-DnaC (yellow) foci per cell before (0 min) and at various times after exposure to UV. Error bars represent SE.

Fig. 3.

Persistence of DNA synthesis after UV. (A) Representative images of a strain carrying ∆yjjG ∆deoB after EdU labeling. Shown is a 2-min pulse of EdU, followed by fixation and coupling of fluorescence through click chemistry. Cells were sampled at various times after UV. (Scale bar: 5 μm.) Contrast and brightness have been normalized. (B) Percentage of cells with at least one focus for EdU before (0 min), and at various times after, UV. Error bars represent SE. (C) Estimated normalized DNA synthesis at various times after UV (Observed), as measured by the integrated intensity of all spots in a cell, compared with the expected synthesis if all remaining DnaB foci in Figs. 1 and 2 were fully functional replisomes (Expected), and with the expected synthesis in cells with fully functional replisomes and no replisome disassembly (Simulated unperturbed). Error bars represent SE. (D) Mean rate per replisome obtained by renormalizing the “Observed” data using the “Expected” data in C. The estimated rates in bp⋅s⁻¹ are shown as reference.

Fig. 4.

Pol III HE recruitment is independent of ongoing replication and dependent on the χ subunit. (A, Left) Description of the system to stop initiation of DNA replication. IPTG-inducible CRISPR-dCas targets the oriC to prevent new replication round. (A, Right) Cells were induced for 2 h. Average number of foci per cell of YPet-DnaB and ε-YPet before and after treatment with UV 5 min after exposure in conditions where initiation proceeds unimpeded (−IPTG) or when it is blocked (+IPTG). (B) Representative images of cells carrying YPet-DnaB or ε-YPet and the oriC blocking CRISPR-dCas system. Arrows mark positions of fluorescent foci. (Scale bar: 1 μm.) (C) Cells were incubated at the restrictive temperature for 2 h. Shown are the average number of foci per cell of YPet-DnaB and ε-YPet in a dnaC2 temperature-sensitive background at permissive and nonpermissive temperature. (D) Representative images of cells carrying YPet-DnaB or ε-YPet in a dnaC2 temperature-sensitive background at nonpermissive temperature. (Scale bar: 2 μm.) (E, Left) Description of the degron system used in this work. (E, Right) Average number of foci per cell of YPet-DnaB and ε-YPet before and after treatment with UV 5 min after exposure in conditions where χ is present (−Ara) or after χ has been degraded (+Ara). (F) Representative images of cells carrying YPet-DnaB or ε-YPet and a degradable copy of the χ subunit. (Scale bar: 1 μm.)

Fig. 5.

UV irradiation affects the dynamics of the replisome. (A) Representative images of ε-mMaple in an sptPALM experiment to characterize its binding kinetics. (Scale bar: 1 μm.) (B) Representative examples of the distribution of fluorescent foci lifespans (blue bars) for Pol III ε subunit, before and after UV, showing fitting of a single-exponential decay model (purple line), the estimated bleaching rate in the same conditions (yellow line), and the corrected estimated bound time (red line). PDF, probability density function. Numbers indicate bound time in seconds with the SE in parenthesis. (Left) Pol III ε subunit before UV. (Right) Pol III ε subunit after UV (C) The distribution of the logarithm of the apparent diffusion coefficient (blue bars) for Pol III ε subunit and LacI bound control (gray bars), before and after UV, showing the fitting of a Gaussian mixture model (red and gray line). The percentage indicates the proportion of diffusing molecules. (Left) Pol III ε subunit before UV. (Right) Pol III ε subunit after UV. The y axis represents probability density function. (D) Model for the replisome slowdown. After encountering an UV lesion on the leading strand, the helicase continues unwinding through this site, but the Pol III is stalled, leading to the uncoupling between the Pol III HE and the helicase. This, in turn, results in a decrease translocation rate of the helicase. Priming by DnaG downstream of the lesion on the leading strand leads to the recruitment of a new copy or the same copy of Pol III to resume DNA synthesis, causing replisome lesion skipping.