activity slowdown after exposure to ultraviolet light in Escherichia coli

Nicolas Soubrya, Andrea Wanga, and Rodrigo Reyes-Lamothea,1

aDepartment of Biology, McGill University, Montreal, QC H3G 0B1, Canada

Edited by Graham C. Walker, Massachusetts Institute of Technology, Cambridge, MA, and approved April 29, 2019 (received for review November 9, 2018) The replisome is a multiprotein machine that is responsible for The effect of UV-C light (190 to 290 nm) on DNA and DNA replicating DNA. During active DNA synthesis, the replisome replication has been widely studied. UV-C generates cyclobutane tightly associates with DNA. In contrast, after DNA damage, the pyrimidine dimers (CPDs) and, to a lesser extent, 6-4 pyrimidine replisome may disassemble, exposing DNA to breaks and threat- adducts (6-4 PAs) on DNA (16). CPDs have been shown to in- ening cell survival. Using live cell imaging, we studied the effect of hibit the activity of Pol III (17) but should allow progression of UV light on the replisome of Escherichia coli. Surprisingly, our the DnaB as they fit through its central pore (18). results showed an increase in Pol III holoenzyme (Pol III HE) foci Shortly after UV treatment, the rate of DNA replication sig- post-UV that do not colocalize with the DnaB helicase. Formation nificantly drops in E. coli, an effect lasting for tens of minutes of these foci is independent of active replication forks and depen- (19, 20). The reduced DNA synthesis is produced in short frag- dent on the presence of the χ subunit of the clamp loader, sug- ments on both strands (21, 22), which was classically interpreted gesting recruitment of Pol III HE at sites of DNA repair. Our results as the result of Pol III hopping over CPDs (21). In agreement also showed a decrease of DnaB helicase foci per cell after UV, with this model, DnaG was reported to prime both strands ahead consistent with the disassembly of a fraction of the . of a lesion that blocks Pol III (23, 24), providing a mechanism by By labeling newly synthesized DNA, we demonstrated that a drop which continued progression of the DnaB helicase would medi- in the rate of synthesis is not explained by replisome disassembly ate recruitment of Pol III HE downstream of the lesions, ef- alone. Instead, we show that most replisomes continue synthe- fectively resulting in lesion hopping. However, previous works sizing DNA at a slower rate after UV. We propose that the slow- that reported replisome stalling shortly after a lesion on the down in replisome activity is a strategy to prevent clashes with leading strand have not yet been fully reconciled with these data BIOCHEMISTRY engaged DNA repair proteins and preserve the integrity of the (25–27). In addition, different live cell studies have led to op- replication fork. posing conclusions on the fate of the replisome, suggesting either stable binding of the DnaB helicase or the complete disassembly DNA replication | replisome | fluorescence microscopy | UV | of the replisome after UV (28, 29). Escherichia coli We used fluorescence microscopy to directly test the effect of UV on the replisome and DNA synthesis at a single-cell level. NA replication is carried out by a multiprotein machine We used slow-growth conditions, with generation times above Dcalled the replisome. In Escherichia coli, the replisome is 100 min, to minimize convolution of our data by new rounds of composed of the DnaB helicase, the DnaG , the DNA DNA replication, as reported previously (29). Our results lead us Pol III (αeθ), the factor β clamp (β2), the clamp to conclude that most replisomes remain active, although pro- loader (τ3δδ′ψχ), and the single strand binding protein SSB (Fig. gressing at a diminished rate, after exposing cells to UV at doses 1A) (1, 2). Multiple protein–protein interactions exist among where CPDs decorate DNA at every few kilobases. Our data are these subcomplexes. DnaB and DnaG interact with each other consistent with the capacity of the replisome to skip over lesions (3, 4). Pol III and clamp loader are physically coupled by the interaction between α and τ, forming the Pol III* subcomplex Significance (5). Pol III* and β clamp form the Pol III holoenzyme (Pol III HE) (6). Finally, the τ subunit mediates the interaction between A multicomponent molecular machine, called the replisome, DnaB helicase and Pol III HE (7). Once in every cell cycle, DNA mediates high fidelity genome duplication but requires error- replication is initiated with the assembly of two replisomes in free DNA as its template. The presence of lesions on DNA, like opposite orientations at a specific locus of the chromosome, the those generated after UV, halt replisome activity and can lead oriC. Each replication fork duplicates half of the 4.6-Mbp cir- to replisome disassembly, a potentially life-threatening situa- −1 cular chromosome at rates between 0.6 and 1 kbp·s at 37 °C tion to the cell. Here, we show that, in cells exposed to UV, (8), completing replication as the two of them meet at the region parts of the replisome independently localize at sites far from opposite from oriC. the place of DNA synthesis. In addition, we provide evidence Successful genome duplication is dependent on undamaged that the replisome responds to UV by slowing down its rate of template DNA. Modifications in the chemistry of DNA and the synthesis on the damaged DNA template. Modulation of the presence of strand discontinuities can inhibit the progression of rate of DNA replication may be a general strategy used by the replisome or lead to its disassembly, potentially generating other organisms to minimize the impact of DNA damage on the double strand DNA (dsDNA) breaks and life-threatening con- duplicating genome. sequences to the cell (9). Specialized DNA repair systems con- tinually act on lesions to restore DNA (10–12). Prolonged Author contributions: N.S. and R.R.-L. designed research; N.S. and A.W. performed re- replisome stalling and replisome disassembly trigger the acti- search; N.S., A.W., and R.R.-L. analyzed data; and N.S. and R.R.-L. wrote the paper. vation of specialized mechanisms that correct dsDNA breaks The authors declare no conflict of interest. and restore the replication fork—theDNAstructureonwhich This article is a PNAS Direct Submission. the replisome acts—followedbymechanismstoreassemble Published under the PNAS license. replisome at those sites (13–15). The frequency at which the 1To whom correspondence may be addressed. Email: [email protected]. replisome disassembles duringnormalgrowthandthetim- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. ing required for replisome reassembly have not been well- 1073/pnas.1819297116/-/DCSupplemental. established.

www.pnas.org/cgi/doi/10.1073/pnas.1819297116 PNAS Latest Articles | 1of7 Downloaded by guest on September 27, 2021 terized E. coli strains carrying derivatives of replisome compo- nents fused to the yellow fluorescent protein YPet (30, 31). We grew cells in conditions where they undergo a single replication event and have at most two replication forks, which translate into cells with zero, one, or two fluorescent spots for DnaB and the proof-reading exonuclease subunit of Pol III, e (Fig. 1B) (30). Five minutes after exposing cells to UV at a dose of 25 J/m2,we observed a small decrease in the average number of YPet-DnaB foci, going from 1.57 ± 0.02 (SE) before to 1.39 ± 0.03 spots per cell after treatment (Figs. 1B and 2D). Disappearance of DnaB spots supports the idea that the replisome has disassembled in those cells. In unexpected contrast, e-YPet showed an increase in the number of spots per cell, going from 1.64 ± 0.03 before to 2.10 ± 0.03 spots per cell after treatment. Notably, this included a significant increase in the percentage of cells with three and four spots, from 8.4 to 29.5% (Fig. 1D). The effects of UV on the distribution of DnaB and e spots correlated with the UV dose used (Fig. 1C). Further characterization of replisome subunits corroborated the existence of two different behaviors in the subcomplexes of the replisome. YPet-DnaG showed a slight reduction in the number of fluorescent spots per cell, similar to DnaB but to a lower extent, going from 1.61 ± 0.04 to 1.52 ± 0.05 before and after treatment, respectively. Whereas reminiscent of the results for e, the number of fluorescent spots increased after UV for β-clamp from 1.9 ± 0.03 to 2.53 ± 0.08; for τ from 1.59 ± 0.04 to 2.06 ± 0.03; for χ from 1.55 ± 0.02 to 2.19 ± 0.04; and for SSB from 1.57 ± 0.03 to 2.40 ± 0.07 (Fig. 1E and SI Appendix, Fig. S3A). These results match the architecture of the replisome as DnaG needs to interact with DnaB for recruitment to the rep- lication fork, and β-clamp, clamp loader, and Pol III form the Pol III HE (Fig. 1A). In addition, our data agree with studies on the replisome dynamics where we and others showed that Pol III and the clamp loader frequently unbind from the replication fork, while the DnaB helicase is stably bound (32, 33). To characterize the two binding regimes in the replisome, we imaged cells carrying both YPet-DnaB and e-mTagRFP (Fig. 2). In untreated cells, we expected to observe colocalizing foci for both subunits as the replisome should contain both of them. Our Fig. 1. Unbinding of replisome subunits at a UV lesion. (A) Model of the results confirm this expectation: The median distance between architecture of an active replisome. DNA is unwound by the DnaB helicase. DnaB and the closest e spot was 0.151 μm (Fig. 2 A and F). In DnaG primase binds to DnaB. Pol III and the clamp loader bind each other agreement with the results described above, the number of DnaB through the τ subunit of the clamp loader, which also mediates binding to β spots decreased and the number of e spots increased after UV the DnaB helicase. The -clamp is left behind the fork after the completion C D SI Appendix B of an Okazaki fragment at the lagging strand. SSB covers ssDNA produced (Fig. 2 and and , Fig. S3 ). The greatest contrast during the cycle of lagging strand synthesis. (B) Representative images of in the spot distribution of these two subunits was reached 30 min cells carrying YPet-DnaB or e-YPet before, and 5 min after, exposure to 25 J/m2 after UV exposure, after which we observed a trend of recovery UV dose. White arrows mark the location of foci. (Scale bar: 1 μm.) (C)Average toward the pretreated state (Fig. 2 C and D). Despite the dif- number of the foci per cell of YPet-DnaB or e-YPet 5 min after exposure to ference in abundance, the distance between DnaB and the different UV doses. (D) Distribution of the number of foci per cell in cells closest e spot did not dramatically change, having a median of e carrying YPet-DnaB or -YPet 5 min after exposure to UV. (E) Average number 0.178 μm after 30 min. Therefore, our results suggest that all of foci for all of the replisome subunits tested: YPet-DnaB, YPet-DnaG, YPet-β, e τ χ replisome subunits remain in the proximity of the replication -YPet, -YPet, -YPet, and SSB-YPet. Pictures were taken 5 min after exposure. — Error bars represent SE. fork after UV treatment although they may not be active as they might be binding to gaps behind the replication fork. They also suggest the recruitment of additional copies of the Pol III on DNA. In addition, we show that the Pol III HE can act in- HE and SSB at other sites on the chromosome, perhaps par- dependently of the DnaB helicase and the replication fork after ticipating in DNA repair or as a result of DNA processing caused being recruited to other sites of the chromosome in a manner by UV lesions. dependent of the χ subunit. Pol III HE activity outside the fork Slowdown in the Rate of DNA Synthesis Is Only Partially Explained by may contribute to filling DNA gaps and, in conjunction with its Replisome Disassembly. Our initial estimates of replisome disas- β -clamp loading activity, may influence other processes like sembly, as measured by the disappearance of YPet-DnaB foci, translesion DNA synthesis. did not account for the elongation of cells after UV. In UV- treated cells, cell division is inhibited, but initiation of DNA Results replication continues (29). To obtain an accurate assessment of Pol III Holoenzyme Is Recruited to Multiple Sites in the Nucleoid after the frequency of replisome disassembly, we simulated the Exposure to UV Light. To test directly for replisome subunit sta- expected number of replisomes per cell in a scenario where no bility after UV treatment, we used fluorescence microscopy in disassembly occurs, making the assumption that UV inhibits cell live cells. We studied the replisome by using previously charac- division but that the rate initiation of DNA replication remains

2of7 | www.pnas.org/cgi/doi/10.1073/pnas.1819297116 Soubry et al. Downloaded by guest on September 27, 2021 To complement these observations, we monitored DnaB reloading using a mNeonGreen fusion to the reloading protein PriA (Fig. 2F and SI Appendix, Fig. S3B). PriA is a crucial component of the protein complex that loads the helicase-loader DnaC-DnaB helicase in an oriC-independent manner (34). The fraction of cells with PriA spots increased after UV treatment from 7 ± 0.2% (SE), and an average of 0.067 ± 0.021 spots per cell, in untreated cells, to 78.9 ± 0.5%, and an average of 1 ± 0.05 spots per cell, 60 min after UV treatment (Fig. 2F), closely matching the estimated replisome loss. We observed a similar trend for cells carrying a YPet derivative of the helicase loader DnaC where the frequency of cells with spots increased after UV treatment from 14.20 ± 3.1% (SE), and an average of 0.19 ± 0.05 spots per cell, in untreated cells, to 61.61 ± 5%, and an average of 0.92 ± 0.03 spots per cell, 60 min after UV treatment (Fig. 2F and SI Appendix, Fig. S3B). However, these results reported a lower frequency of cells with DnaC foci compared with PriA, despite the role of DnaC in both oriC-dependent and -independent initiation. This may suggest a significant time delay between PriA binding and DnaC recruitment during reloading of the replisome. We then estimated the rate of DNA synthesis at a single cell level by fluorescently labeling newly replicated DNA through a 2-min pulse of ethyl deoxyuridine (EdU) and azide-coupled Fluor 545 (35). The number of cells with EdU fluorescent foci dropped slightly, from 93.8 ± 0.05 (SE) before UV to 82.5 ± 8.4 after 30 min (Fig. 3 A and B), indicating that DNA synthesis

continued in most cells after UV treatment. We estimated the BIOCHEMISTRY rate of new DNA incorporation based on the integrated intensity of detected spots in a given cell. DNA synthesis sharply de- creased after 5 min of UV treatment, resulting in only 16.2 ± 5.8% (SE) of the incorporation in untreated cells, and pro- gressively increasing, reaching 81.9 ± 13.8% of the original incorporation after 60 min (Fig. 3C, Observed). This decrease in DNA synthesis could be due to the fewer number of replisomes in cells following UV-induced replisome disassembly. To test this hypothesis, we calculated the predicted rate of synthesis based on our estimates of replisome loss, assuming unimpeded DNA synthesis with the same constant rate as before treatment (Fig. 3C, Expected). Interestingly, replisome loss could only account for a small part of the observed decrease in DNA synthesis, raising the possibility that the remaining replisomes act more slowly after UV. We used this “expected” rate to normalize the observed EdU incorporation so we could assess the relative synthesis rate per replisome (Fig. 3D). The corrected rates of synthesis showed that replisomes progress at 18.7% and 84.7% of Fig. 2. DnaB remains in proximity to Pol III after UV. (A) Representative images of a strain carrying YPet-DnaB and e-mTagRFP before, and at various times after, their unperturbed rate 5 and 60 min after UV, respectively. Since UV treatment. White arrows mark colocalization of foci in the two channels. replicating the entire chromosomes takes 65 min at 37 °C (30), we −1 (Scale bar: 1 μm.) (B) Distribution of apparent distances between a YPet-DnaB estimate that the rate of replication goes from ∼600 bp·s before −1 focus and the closest e-mTagRFP focus in a cell. The untreated sample is com- to ∼114 bp·s 5 min after UV. These results show that a consid- pared with the results at 10, 30, and 60 min after UV. The median (M) of each erable proportion of replication forks progress at a diminished population is shown. (C) Average number of foci per cell for YPet-DnaB and average rate after UV treatment. e-mTagRFP before (0 min) and at various times after treatment. (D) Number of e cells with at least one fluorescent focus for YPet-DnaB or -mTagRFP at various Recruitment of the Pol III HE Does Not Require Active Replication. times after treatment. (E) Estimation of helicase disassembly at various times Copies of Pol III HE subunits at sites outside of the replication after UV treatment. (Inset) Numbers are based on the difference between a fork may mark points of DNA repair. Alternatively, they may simulated growth of foci (green line), assuming no dissasembly, and the data of oriC YPet-DnaB from Fig. 1C and C (yellow line). (F) Number of PriA-mNeonGreen represent new DNA replication events from or mark sites (green) and YPet-DnaC (yellow) foci per cell before (0 min) and at various times behind the replication fork. To test these later models, we tar- after exposure to UV. Error bars represent SE. geted an inactive copy of Cas9 (dCas9) to oriC, which has pre- viously shown to inhibit initiation of DNA replication (36) (SI Appendix, Fig. S1B). We then waited for a period of 2 h to allow unchanged (29), resulting in an increase in the number of completion of ongoing DNA replication events. In these condi- replisomes per cell over time (Fig. 2E). Comparing our results tions, we observed a relatively high background of DnaB spots. with the simulated data showed that replisome disassembly We speculate that this is because the inhibition of initiation by continued for about 30 min, plateauing at an average of dCas9 may still permit binding, but not activation, of DnaB at the 0.8 replisomes lost per cell, when >30% of the replisomes are oriC. But consistent with the results above, the number of spots lost (Fig. 2E). Recovery of replisomes is not observed even after in arrested cells did not change substantially after UV, going 60 min. from 0.64 ± 0.05 to 0.58 ± 0.07 (Fig. 4 A and B). In contrast, the

Soubry et al. PNAS Latest Articles | 3of7 Downloaded by guest on September 27, 2021 Recruitment of the Pol III HE Outside the Replication Fork Depends on the χ Subunit of the Clamp Loader. Pol III HE interacts with the DnaB helicase at the replication fork, but our data suggested that it can also be at other sites independently of DnaB. To explain this observation, we hypothesized that SSB serves as an anchor for the Pol III HE at these extra sites as this protein binds to ssDNA, and SSB interacts with the χ subunit of the clamp loader (37). We tested this idea by inducing rapid depletion of the χ subunit in a strain carrying a degron-tagged version of this protein (Fig. 4 E and F) (31, 38). Imaging was done 45 min after arabinose induction. We observed a sharp decrease in the number of e-YPet spots after UV, going from 1.22 ± 0.04 before to 0.63 ± 0.05 spots per cell after UV (Fig. 4 E and F). Depletion of χ had a smaller effect on YPet-DnaB after UV where the number of foci decreased from 1.39 ± 0.04 to 1 ± 0.04, showing a similar trend as in conditions where χ is present (Fig. 4 E and F). These results are consistent with the idea that χ helps to recruit Pol III HE at other sites of the chromosome. Nonetheless, the χ subunit has also been reported to have a role in replisome sta- bility (39). Consequently, an alternative interpretation of these results is that UV further destabilizes the weakened χ-depleted replisome. Indeed, we observed a drop in e-YPet foci in χ-depleted conditions before UV, compared with control cells (Fig. 4 E and F), which agrees with a role for χ in retaining Pol III HE at the fork. Consequently, at present, we cannot rule out a contribution of the stabilizing role of χ in our results.

Exposure to UV Light Changes the Binding Dynamics of the Pol III HE. To further characterize the role of the additional Pol III HE spots in the cell, we studied the binding kinetics of the DNA Pol III using single-molecule experiments. We used a fusion of e with the photoconvertible fluorescent protein mMaple (32, 40) and Fig. 3. Persistence of DNA synthesis after UV. (A) Representative images of the technique of single-particle tracking Photoactivated Locali- a strain carrying ΔyjjG ΔdeoB after EdU labeling. Shown is a 2-min pulse of zation Microscopy (sptPALM) to determine the average resi- EdU, followed by fixation and coupling of fluorescence through click dence time (bound time) of this subunit, as previously described chemistry. Cells were sampled at various times after UV. (Scale bar: 5 μm.) (Fig. 5A) (32). Between 15 and 30 min after UV, we observed an Contrast and brightness have been normalized. (B) Percentage of cells with increase in the bound time going from 10.80 s ± 0.94 before at least one focus for EdU before (0 min), and at various times after, UV. treatment, consistent with previous results (32), to 16.18 s ± Error bars represent SE. (C) Estimated normalized DNA synthesis at various B times after UV (Observed), as measured by the integrated intensity of all 2.11 after treatment (Fig. 5 ). These results are reminiscent of spots in a cell, compared with the expected synthesis if all remaining DnaB the increased bound time observed for Pol III* after treatment foci in Figs. 1 and 2 were fully functional replisomes (Expected), and with the with the DNA inhibitor hydroxyurea (32). expected synthesis in cells with fully functional replisomes and no replisome We also determined the proportion of e bound to DNA before disassembly (Simulated unperturbed). Error bars represent SE. (D) Mean rate and after UV by sptPALM. Capturing pictures at 20-ms rates per replisome obtained by renormalizing the “Observed” data using the under low-intensity continuous 405-nm laser activation, resulted −1 “Expected” data in C. The estimated rates in bp·s are shown as reference. on average in a single fluorescent spot per cell per frame. We characterized the behavior of DNA-bound molecules by studying a strain carrying LacI-mMaple and a lacO array where most of number of e-YPet foci went from 0.29 ± 0.02 to 1.25 ± 0.08 after C A B the molecules are bound to the chromosome (Fig. 5 ) (32). The UV in the induced cells (Fig. 4 and ). As an independent distribution of apparent diffusion coefficients for e-mMaple be- confirmation of these results, we used previously characterized fore treatment showed at least two fractions, one of which rep- strains carrying a dnaC2 temperature-sensitive allele to block resented the DNA-bound molecules—overlapping with the LacI initiation of DNA replication (30). As before, we allowed com- data—and a second showing the diffusive fraction (Fig. 5C). pletion of ongoing replication events by incubating cells at the Using a Gaussian mixture model to estimate the fraction of restrictive temperature (37 °C) for 2 h. Similar to the results DNA-bound molecules in the population, we estimated that the above, we observed a relatively high background of DnaB spots, proportion increased from 16.4% before treatment to 35.5% but, consistent with previous experiments, the number of spots in between 15 and 30 min after UV treatment (Fig. 5C). These data arrested cells changed only slightly after UV, from 0.87 ± 0.08 to support the idea that the Pol III HE bound to DNA is not always 0.98 ± 0.05 (Fig. 4 C and D). In contrast, the number of e-YPet active after UV. It also shows that exposure to UV increases the foci went from 0.10 ± 0.02 to 0.80 ± 0.04 after UV when cells length and the frequency of the binding events of Pol III* to DNA. were incubated at the restrictive temperature (Fig. 4 C and D). Thus, we determined that the additional Pol III HE foci are not Discussion due to new initiation events. In addition, our results also dem- We have investigated the fate of the replisome after encoun- onstrate that additional Pol III HE foci can form even in con- tering a DNA lesion capable of inhibiting Pol III activity but that, ditions where there are no replication forks. Hence, even if some in principle, does not threaten DnaB helicase integrity. A large of these foci may be produced by Pol III HE binding behind the body of literature that studied the effect of UV on DNA repli- replication fork during active synthesis, a fraction of them are cation, spanning the last 50 y, precedes our work. However, this likely to bind elsewhere in the chromosome. report focuses on the effect of UV on the replisome subunits,

4of7 | www.pnas.org/cgi/doi/10.1073/pnas.1819297116 Soubry et al. Downloaded by guest on September 27, 2021 χ Fig. 4. Pol III HE recruitment is independent of ongoing replication and dependent on the subunit. (A, Left) Description of the system to stop initiation of BIOCHEMISTRY 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 e-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 e-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 e-YPet in a dnaC2 temperature-sensitive background at permissive and nonpermissive temperature. (D) Representative images of cells carrying YPet-DnaB or e-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 e-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 e-YPet and a degradable copy of the χ subunit. (Scale bar: 1 μm.)

resulting in unique insight into the response of the replisome to after UV even when cells are not replicating (Fig. 4 A–D), our DNA damage and the dynamics of DNA synthesis on a damaged data suggest that there are other recruitment sites in the chro- DNA template. We expect that our conclusions will be also valid mosome which are independent of the replication fork. Loading to other nonbulky DNA modifications that do not hinder of β-clamp away from the DNA replication fork and subsequent helicase translocation. unbinding of the clamp loader would agree with a lack of overlap Contrary to the long-held belief that the replisome subunits in the localization of the clamp loader and the translesion remain bound during long periods of the replication cycle, recent Pol IV and Pol V during DNA repair (44–46) al- data from our group and other groups have demonstrated much though we note that the presence of the Pol III HE at those sites more frequent turnover in this complex. Single-molecule in vivo may facilitate resuming DNA synthesis after bypass by Pol V. data from Bacillus subtilis and E. coli have shown that the active replicative DNA polymerase is replaced every few seconds (32, A Fast-Acting Rate Switch Strategy as Response to DNA Damage. A 41). Similar observations were made in an independent study much clearer advantage of a dynamic Pol III* binding is an al- using a reconstituted E. coli replisome in vitro (33). Replisome most immediate slowdown of the rate of DNA replication in disassembly exposes DNA structures sensitive to breakage, which response to DNA damage. Such a strategy would reduce the can occur independently of external aggressions such as UV. It is probability of encounters between the DnaB helicase and the therefore unclear why a stable replisome complex was not fa- engaged NER system, which may lead to helicase disassembly. It vored in evolution. is unlikely that the rate change is due to competition of Pol III Our work unveils potential advantages of a dynamic repli- with the translesion polymerases as this effect should increase some. The data are consistent with the recruitment of Pol III HE with the induction of the SOS response and should be minimal at to sites other than the replication fork in a DnaB-independent 5 min after UV. Instead, we propose that Pol III disengages from manner. This is a striking result as these two subcomplexes were DnaB after encountering a CPD, triggering a change in DnaB assumed to be found only at the replisome. We favor the model translocation rate (Fig. 5D). Previous reports show slower DnaB that Pol III HE is recruited at ssDNA gaps, given the presence of helicase translocation in the absence of Pol III* (47, 48). Our SSB. Polymerization activity at these sites is also likely and results show that, shortly after treatment with UV, the replisome − agrees with reports that established Pol III activity at ssDNA proceeds at about 114 bp·s 1 (30) (Fig. 3D). This is just slightly − gaps (42, 43). Pol III HE might also contribute to loading of higher than the ∼80 bp·s 1 single-molecule estimates of the β-clamp at these sites, which could then serve for the recruitment DnaB helicase activity when it uncouples from DNA synthesis of Pol V translesion polymerase. Some of these sites are likely to (49, 50). Thousands of CPDs are generated in the chromosome be found behind the replication fork, resulting from the repli- after a 25 J/m2 UV dose—we estimate one every ∼9 kbp on the some skipping over UV lesions. But since Pol III HE foci formed leading strand based on Courcelle et al. (51). The rate of DNA

Soubry et al. PNAS Latest Articles | 5of7 Downloaded by guest on September 27, 2021 DNA replication averages described above using 2-min pulses of EdU labeling would then be the result of multiple cycles of engagement and disengagement, each lasting a few seconds. As such, the rate of replisome progression reflects the density of lesions on DNA. A much greater number of replisomes simultaneously acting on the genome in eukaryotic cells, although progressing at slower rates, should increase the rate of clashes with the DNA repair systems. As in bacteria, a mechanism to rapidly modulate the rate of replisome progression has been proposed. Binding of the Mrc1–Tof1–Csm3 (MTC) complex in budding yeast has been shown to be needed for a maximal rate of DNA synthesis (53–55). In addition, the interaction between the replisome and MTC is highly dynamic, making it a good candidate to mediate a fast response to DNA damage (56). As such, fast modulation of DNA synthesis by the replisome seems to be a common strategy evolved in both eukaryotes and prokaryotes. It is tempting to speculate that replisome slowdown in eukary- otes precedes the global DNA damage response (DDR), mediated by protein kinases, which should take more time to be established (57).

Continued DNA Replication after UV Supports a Replisome Lesion Skipping Model. A classical model suggests that, after UV, DNA Pol III can hop on DNA to avoid prolonged stalling at lesions (21). A mechanistic explanation was provided by data showing that helicase progression provides a platform for prim- ing on both strands, mediating polymerase lesion skipping after UV treatment (23, 24). We note that the original envisioning of this model, where the same copy of the DNA polymerase re- sumes synthesis after skipping a DNA lesion, is unlikely to occur in cells, given that Pol III* is frequently exchanged (see above). Hence, it is more probable that the resumption of DNA synthesis occurs after a different copy of Pol III* is recruited from the diffusing pool. However, consistent with the idea of lesion skipping by the replisome, our data provide yet another set of evidence by showing that, even though the rate of DNA synthesis drops after UV exposure, DNA synthesis continues (Fig. 5D). Previous reports were not able to distinguish between replisome slowdown, disassembly, and cell heterogeneity as they were based on population averages. Furthermore, we show that β-clamp continues to accumulate at sites containing DnaB heli- case (SI Appendix, Fig. S3) and that the DnaB helicase remains Fig. 5. UV irradiation affects the dynamics of the replisome. (A) Repre- in proximity of e at all times after UV (Fig. 2), further supporting sentative images of e-mMaple in an sptPALM experiment to characterize its the idea that the activities of priming and DNA elongation binding kinetics. (Scale bar: 1 μm.) (B) Representative examples of the dis- continue after UV. tribution of fluorescent foci lifespans (blue bars) for Pol III e subunit, before and after UV, showing fitting of a single-exponential decay model (purple Experimental Procedures line), the estimated bleaching rate in the same conditions (yellow line), and the corrected estimated bound time (red line). PDF, probability density Detailed descriptions of the experimental procedures used are available in SI function. Numbers indicate bound time in seconds with the SE in paren- Appendix, Supplementary Experimental Procedures. In brief, strain con- thesis. (Left) Pol III e subunit before UV. (Right) Pol III e subunit after UV (C) struction was done using P1 transduction or lambda red recombination in an The distribution of the logarithm of the apparent diffusion coefficient (blue AB1157 background (58). Cells were routinely grown in Luria broth or in bars) for Pol III e subunit and LacI bound control (gray bars), before and after M9 glycerol. For microscopy, cells were grown overnight in M9 glycerol and UV, showing the fitting of a Gaussian mixture model (red and gray line). then diluted in the same medium and grown to an OD600 between 0.1 and The percentage indicates the proportion of diffusing molecules. (Left)Pol 0.2. For degron experiments, cells were induced with 0.5% arabinose at an III e subunit before UV. (Right)PolIIIe subunit after UV. The y axis rep- OD600 of 0.1 after the dilution in M9 and grown for 45 to 60 min before resents probability density function. (D) Model for the replisome slow- imaging. For degron CRISPR experiments, cells were induced with 0.5 mM β down. After encountering an UV lesion on the leading strand, the helicase isopropyl -D-1-thiogalactopyranoside (IPTG) 45 min after the dilution in continues unwinding through this site, but the Pol III is stalled, leading to M9 and grown for 2 h before imaging. DnaC2 strains were incubated at the uncoupling between the Pol III HE and the helicase. This, in turn, re- 37 °C for 2 h before UV treatment. Cells were spotted on a 1% agarose pad sults in a decrease translocation rate of the helicase. Priming by DnaG in M9 glycerol. UV irradiation was performed in a UV Stratalinker 2400 downstream of the lesion on the leading strand leads to the recruitment of (Stratagene) at the dose specified in the text before placing the coverslip. a new copy or the same copy of Pol III to resume DNA synthesis, causing Images were taken 5 min after irradiation unless stated otherwise. For replisome lesion skipping. single-molecule experiments, collection of images started at 15 min and ended at 30 min postirradiation. Imaging was performed at room temper- ature on an inverted Olympus IX83 microscope from a single-line cellTIRF > illuminator (Olympus). For EdU incorporation, all cells were grown overnight synthesis progressively increased, reaching 80% of the orig- in M9 glycerol at 37 °C and then diluted in the same medium and grown to

inal rate 60 min after UV, in agreement with the proposed an OD600 between 0.1 and 0.2. A sample of cells was put aside as an un- dynamics of CPD removal after exposure to UV (52). The treated control while the rest of the liquid culture was put in a sterile Petri

6of7 | www.pnas.org/cgi/doi/10.1073/pnas.1819297116 Soubry et al. Downloaded by guest on September 27, 2021 dish and then UV irradiated. EdU to a concentration of 20 μg/mL was added ACKNOWLEDGMENTS. We thank Anjana Badrinarayanan, Gregory Marczynski, for 2 min before fixation at specified times after UV exposure. Fixing and Christian Rudolph, Steph Weber, and members of the R.R.-L. laboratory for labeling were done using a modified protocol described in ref. 59. All helpful discussions. Some of the experiments were done using equipment analysis was done using custom Matlab scripts and TrackMate software in from the Integrated Quantitative Bioscience Initiative (IQBI), funded by Canada Foundation for Innovation 9. This work was funded by the Natural ImageJ. A custom Matlab script was used to simulate the increase in the Sciences and Engineering Research Council of Canada (NSERC 435521- number of copies of replisomes per cell when cell division is inhibited and 2013), the Fonds de Recherche du Quebec–Nature et Technologies (FRQNT replication proceeds at a slower rate. The number of repeats, sample size, 2017-NC-198919), the Canadian Institutes for Health Research (CIHR MOP and statistical tests for pairwise comparisons for all microscopy analysis 142473), the Canada Foundation for Innovation (CFI 228994), and the Canada presented in the figures can be found in SI Appendix, Table S4. Research Chairs program.

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