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Characterization of a pathway of genomic instability induced by R-loops and its regulation by topoisomerases in E. coli [1]

['Julien Brochu', 'Département De Microbiologie', 'Infectiologie Et Immunologie', 'Université De Montréal', 'Montréal', 'Émilie Vlachos-Breton', 'Dina Irsenco', 'Marc Drolet']

Date: 2023-06

The prototype enzymes of the ubiquitous type IA topoisomerases (topos) family are Escherichia coli topo I (topA) and topo III (topB). Topo I shows preference for relaxation of negative supercoiling and topo III for decatenation. However, as they could act as backups for each other or even share functions, strains lacking both enzymes must be used to reveal the roles of type IA enzymes in genome maintenance. Recently, marker frequency analysis (MFA) of genomic DNA from topA topB null mutants revealed a major RNase HI-sensitive DNA peak bordered by Ter/Tus barriers, sites of replication fork fusion and termination in the chromosome terminus region (Ter). Here, flow cytometry for R-loop-dependent replication (RLDR), MFA, R-loop detection with S9.6 antibodies, and microscopy were used to further characterize the mechanism and consequences of over-replication in Ter. It is shown that the Ter peak is not due to the presence of a strong origin for RLDR in Ter region; instead RLDR, which is partly inhibited by the backtracking-resistant rpoB*35 mutation, appears to contribute indirectly to Ter over-replication. The data suggest that RLDR from multiple sites on the chromosome increases the number of replication forks trapped at Ter/Tus barriers which leads to RecA-dependent DNA amplification in Ter and to a chromosome segregation defect. Overproducing topo IV, the main cellular decatenase, does not inhibit RLDR or Ter over-replication but corrects the chromosome segregation defect. Furthermore, our data suggest that the inhibition of RLDR by topo I does not require its C-terminal-mediated interaction with RNA polymerase. Overall, our data reveal a pathway of genomic instability triggered by R-loops and its regulation by various topos activities at different steps.

DNA topoisomerases act during replication, transcription, and recombination to solve topological problems inherent to the double-helical structure of DNA. Topos of the type 1A family are the only ones that are ubiquitous. The prototype enzymes of the two main type IA subfamilies are topo I and topo III from Escherichia coli. The recent finding that E. coli topo I and III can suppress R-loop formation suggested that R-loops have been a problem early in the evolution of life. However, how toxic R-loops are generated and how they exert their detrimental effect on genome stability, especially in the absence of type IA topos, is still largely unknown. Here, we have uncovered a pathway leading to genome instability in the absence of type IA topos: unregulated replication from R-loops involving RNAP backtracking leads to DNA amplification in the chromosome terminus region due to replication forks blocked at termination barriers. Furthermore, our data suggest that type IA topos play major roles at the termination step of replication when convergent replication forks merge and R-loops exert their detrimental effects mostly via topological problems. Overall, our data shed new light on how R-loops can lead to genomic instability and how topos can deal with this problem.

Funding: This work was supported by a Discovery Grant from the National Sciences and Engineering Research Council of Canada ( https://www.nserc-crsng.gc.ca/ , RGPIN-2022-03760) to M.D. JB and E.V.-B. are respectively supported by an Alexander Graham Bell Doctoral scholarship from the NSERC ( https://www.nserc-crsng.gc.ca/ ) and a Graduate scholarship form the NSERC. Funding for open access charge: National Sciences and Engineering Research Council of Canada. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Despite much being known about the biochemical properties and main functions of type IA topos in E. coli, much less is known about the consequences of losing their activity on genome stability. Elucidating the roles of type IA topos in genome maintenance is important, as it may reveal new pathways for genomic instability and help explain the conservation of these enzymes through evolution. Moreover, inhibitors of type IA topos are being developed to be used as antibiotics [ 54 ]. The finding that deleting both topA and topB in E. coli generated a new phenotype, suggested that either both enzymes share a common function, can act as backups for each other and/or a combination of defects attributed to the absence of topo I or topo III was responsible for this new phenotype: extensive cellular filamentation with a recA-dependent chromosome segregation defect [ 55 ]. This phenotype was recently correlated with RecA- and R-loop-dependent extensive over-replication in Ter region [ 14 ]. Here, it is shown that the involvement of RLDR is largely indirect and likely related to the accumulation of replication forks reaching Ter/Tus barriers that in turn increases the likelihood of over-replication in Ter. Furthermore, the data suggest that the lack of type IA topos decatenation activity during replication elongation and forks fusion (pathway 2) stimulate over-replication. This causes a high amount of DNA accumulation within the small TerA-TerB interval including dif that leads to a chromosome segregation defect corrected by topo IV overproduction. Overall, our data suggest that the lack of type IA topos may simulate Ter over-replication via excess negative supercoiling (mostly topA) that promotes RLDR and defective processing of forks at Ter/Tus barriers, and via the lack of decatenation activity (mostly topB) during replication elongation and termination.

Experimental evidence has been presented which suggests that type IA topos might be involved in the process of replication termination. In one report, it was shown that excess negative supercoiling caused by a mutation in the C-terminal domain of topo I could reduce the ability of Tus to arrest replication forks [ 53 ]. The process of replication termination when two replication forks converge represents a major topological challenge ( Fig 1B ). Indeed, when the converging forks are about to merge, not only does positive supercoiling build-up to very high levels but the space on the DNA template also becomes too small to accommodate the binding of DNA gyrase molecules. At this stage, two alternative pathways [ 4 , 32 ] can be used to complete replication while allowing the last intertwines to be removed to permit chromosome segregation. In the first major pathway, replication forks rotation allows the positive supercoiling to migrate behind the forks as pre-catenanes that are converted to catenanes once replication is completed. These catenanes are removed by topo IV (Pathway 1). In the second pathway, the parental strands are unlinked by topo III before the completion of replication e.g., by pol I to fill the gaps [ 32 , 38 ]. Whether this pathway is used in E. coli is currently unknown.

(A) Representation of the circular chromosome of E. coli with the oriC region (red dot) from which bidirectional replication is initiated (red arrows). The convergent replication forks normally meet in the region opposite to oriC (dif). TerA to TerJ are polar Ter/Tus barriers that block forks coming from only one direction (indicated by the black arrows). See text for more details. (B) Topological problems associated with replication termination when two replication forks converge. Topo III and topo IV can remove pre-catenanes. In pathway 1 (the main one), replication is fully terminated before the full unlinking of the replicated sister chromosomes by topo IV. In pathway 2, the sister chromosomes are fully unlinked by a type IA topo (mostly topo III) before the completion of the replication. Topo III is shown in green acting at the fork. See text for details.

Bidirectional replication of the circular E. coli chromosome is initiated at oriC and terminated within the opposite broad terminus (Ter) region, where the two convergent replication forks meet and fuse. The final chromosome decatenation step by topo IV and the resolution of chromosome dimers by XerCD take place within this region at the dif site [ 38 ]. The trapping of replication forks by Tus proteins that binds to polar Ter sequences (10 of them, TerA to TerJ, Fig 1A ) restrains the termination process to Ter [ 38 ]. Ter sequences within the left portion of the chromosome block clockwise-moving forks, whereas those in the right portion of the chromosome block counterclockwise-moving forks. Under normal growth conditions, replication forks merge at the position opposite to oriC [ 39 ] or very close to this position, where the clockwise fork is first arrested at TerC [ 38 ]. The process of replication termination is more complex than initially believed in part because fork fusion can lead to over-replication as originally shown in vitro [ 40 ]. In fact, several proteins including RecG, RecBCD, ligase A, Pol I, RNase HI, Exo I, Exo VII, SbcCD, and RecJ nucleases appear to be involved in the processing of replication termination intermediates [ 41 – 50 ]. The inactivation of many of these proteins alone or in combination has been shown to lead to over-replication to various extents in the Ter region. This hallmark phenotype is characterized by the appearance of a prominent Ter peak bordered by the innermost Ter/Tus barrier as shown by MFA by next-generation sequencing (NGS) [ 41 , 42 , 44 – 46 , 48 , 50 , 51 ]. Termination involving a Ter/Tus barrier normally prevents over-replication [ 40 ]. However, when the arrested fork is stuck at the barrier before the arrival of the convergent fork it can lead to over-replication [ 44 , 52 ]. Ter/Tus-dependent and independent Ter over-replication requires RecA for D-loop formation that is used for PriA-dependent primosome assembly [ 41 , 43 , 51 ].

Unlike topA null mutants, topB null cells grow as well as wild-type cells, but show a delayed and disorganized nucleoid segregation phenotype, as compared with wild-type cells [ 31 ]. Topo III is found to be associated with replication forks in vivo (via an interaction with SSB and DnaX; [ 31 , 32 ]), where its substrate, ssDNA, is present. In vitro, topo III has a strong decatenation activity that can fully support replication including the termination step [ 33 ]. Thus, it is very likely that topo III plays a role in the removal of pre-catenanes during replication [ 31 , 34 ]. However, at least when the activity of other topoisomerases is not disturbed, this role appears to be relatively minor compared with topo IV, the absence of which inhibits growth and causes severe chromosome segregation defects (the par phenotype [ 13 ]). In fact, when topo IV [ 35 , 36 ] or gyrase [ 37 ] activity is significantly perturbed, topo III becomes essential for decatenation.

Null mutations in the topA gene of E. coli inhibit cell growth. Compensatory mutations may arise that allow topA null cells to generate visible colonies (reviewed in [ 4 ]). Such mutations are either substitutions in gyrA or gyrB genes that reduce the negative supercoiling activity of gyrase [ 9 , 10 ], or amplification of a chromosomal region allowing topo IV overproduction [ 4 , 11 – 14 ]. DNA gyrase introduces negative supercoiling, via the relaxation of positive supercoiling generated during replication [ 15 ] and transcription [ 16 ]. The main function of topo IV is to act as a decatenase behind the replication fork to remove precatenanes and, once chromosomal replication is completed, to remove catenanes to allow chromosome segregation [ 17 , 18 ]. Evidence of a minor role of topo IV in the regulation of chromosome topology via the relaxation of negative supercoiling has been reported [ 19 ]. Topo I interacts with RNA polymerase (RNAP) [ 20 ] and relaxes negative supercoiling generated behind moving RNAPs during transcription [ 21 ]. The failure to relax transcription-induced negative supercoiling in topA null mutants leads to hypernegative supercoiling and the formation of R-loops that can inhibit gene expression and activates R-loop-dependent replication (RLDR hereafter) [ 22 – 29 ]. Thus, one major function of topo I in E. coli is the relaxation of transcription-induced negative supercoiling to inhibit R-loop formation. Genome-wide approaches have recently been used to demonstrate this major function of topo I [ 30 ].

Topos of the type IA family are the only ones that are ubiquitous [ 3 , 4 ]. They use a strand-passage mechanism to relax or decatenate DNA. Unlike other topos, they use ssDNA as substrates and many of them possess RNA topo activity [ 3 , 5 , 6 ]. Type IA topos are classified into three subfamilies, the two main ones being topo I and topo III with E. coli topA and topB encoding the prototype enzyme of these two subfamilies. Topo I is present in all bacteria but not in archaea and eukaryotes, whereas topo III is found in most bacteria and in all archaea and eukaryotes. Topo III has a higher requirement for ssDNA than topo I and mostly acts as a decatenase [ 7 ], whereas topo I mainly acts to relax negative supercoiling [ 4 ]. The results of recent single-molecule experiments suggest that a dynamic fast gate for topo I may promote efficient relaxation of negatively supercoiled DNA. In contrast, a slower gate-closing for topo III may facilitate capture of dsDNA and, as a result, efficient decatenation [ 8 ].

Because of the double-helical structure of DNA, each time the two strands are separated during replication, transcription, or repair, underwinding and overwinding (supercoiling) occur. Such excess supercoiling, in turn, interferes with normal gene expression and replication. Furthermore, intertwining of the DNA occurs during replication and repair. If not correctly resolved, such intertwining inhibit chromosome segregation and may lead to DNA breaks and genomic instability. To solve these topological problems, cells possess DNA topoisomerases (topos), which are nicking-closing enzymes [ 1 , 2 ], which cut one (type I) or two (type II) DNA strands. Type I and II are further divided respectively into types IA and IB and IIA and IIB. To solve topological problems, type IA and type II enzymes use a strand-passage mechanism whereas enzymes of the type IB family, also named swivelases, use a rotation mechanism.

Results

A deletion approach failed to reveal strong RLDR origins that could be responsible for Ter over-replication in topA topB null cells RLDR was first observed in rnhA null cells as a kind of replication that was resistant to protein synthesis inhibition (constitutive stable DNA replication (cSDR), as opposed to replication from oriC [56,57]. MFA experiments with 150 to 200 kb long hybridization probes initially revealed a peak in Ter region and led the authors to conclude for the presence of origins for RLDR (oriKs) in this region [58]. More recently, in a series of experiments with MFA by NGS, the authors were able to precisely delineate the Ter peak in rnhA null mutants, but no evidence was found for the presence of well-defined RLDR origins in this region [50]. In fact, in a laboratory evolution experiment, the only strong RLDR origin that could support good growth of a rnhA null mutant without the oriC/DNA system, was localized far away from the Ter region [59]. These recent results, together with the finding that Ter over-replication is often caused by defective replication termination, led us to investigate in more details the source of this over-replication in topA topB null mutants. If present, a strong origin (oriK) of bi-directional replication should be located at the top of the Ter peak. The highest copy number identified from the MFA data by NGS for topA topB null cells is in a region close to the genomic position 1.52 [14], where the two divergently transcribed genes yncD and yncE are found. topA topB null strains carrying either a yncD (JB134) or yncE (JB335) deletion were first constructed. The topA and topA topB strains that were used previously and in the present study unless otherwise indicated, carry a gyrB(Ts) allele [14,27–29]. Such strains grow better at 37°C than at 30°C owing to the partial inactivation of GyrB, reducing the negative supercoiling activity of gyrase. Temperature downshifts to 30°C rapidly re-activates gyrase activity which lead to the full expression of topA null phenotypes, including slow growth, hypernegative supercoiling and high level of R-loop formation and RLDR [14,22,27,29]. In our studies, these strains were grown at 30°C (see Material and Methods). Fig 2A shows the MFA profiles by NGS of genomic DNA from JB137 (topA topB) and JB335 (topA topB yncE) strains. Spc means that spectinomycin was added to log phase cells for two hours before genomic DNA extraction as previously done [14]. After two hours, oriC-dependent replication is fully terminated, whereas RLDR is still active. The Ter peak heights, regardless of whether spc was added, were found to be indistinguishable between the strains, indicating that yncE plays no role in Ter over-replication (Fig 2A). As expected, since RLDR was still active, the Ter peak height increased after the spc treatment as previously observed [14]. The ydcM/lepA ratio by qPCR with genomic DNA from JB137 (topA topB), JB134 (topA topB yncD) and JB335 (topA topB yncE) was also determined. ydcM is located close to the top of the Ter peak and lepA is outside Ter, and it was previously shown that the ydcM/lepA ratio accurately reflected the Ter peak height revealed by MFA [14]. Fig 2B shows that the ydcm/lepA ratios were very similar between the strains [14]. Thus, yncE and yncD plays no significant roles in Ter over-replication. PPT PowerPoint slide

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TIFF original image Download: Fig 2. MFA and qPCR show that deleting genes located at the top of the Ter peak in topA topB null cells had no effects on the peak height. (A) MFA by NGS of genomic DNA extracted from JB137 (ΔtopB topA20::Tn10 gyrB(Ts)) and JB335 (ΔtopB ΔyncE topA20::Tn10 gyrB(Ts)) cells grown at 30°C to log phase and treated (spc) or not treated (no spc) with spectinomycin for two hours. The absolute read counts (Log2) were plotted against chromosomal coordinates (W3110 genomic sequence AP009048.1). The gray line is the loess regression curve. amp indicates the amplified parC parE region. The oriC region as well as TerA, TerB and TerG barriers, are shown. The gap at position around 0.3 corresponds to the Δ(codB-lacI)3 deletion in these strains. (B) ydcM/lepA ratio determined by qPCR of genomic DNA extracted from JB137 (ΔtopB topA20::Tn10 gyrB(Ts)), JB335 (ΔtopB ΔyncE topA20::Tn10 gyrB(Ts)), JB631 (ΔtopB ΔydcD topA20::Tn10 gyrB(Ts)) and JB134 (ΔtopB ΔyncD topA20::Tn10 gyrB(Ts)) cells grown at 30°C to log phase as described in Material and Methods. Additional mutation, refers to the additional mutation under study that is also present in the topA topB gyrB(Ts) strain. https://doi.org/10.1371/journal.pgen.1010754.g002 Preliminary DRIP-seq data to map R-loops genome-wide in topA topB null mutants revealed a good candidate for a possible oriK, close to the top of the Ter peak. R-loop formation in topA topB null cells at this site, within ydcD (close to 1.53), was confirmed by DRIP-qPCR as shown in S1 Fig. The topA topB ydcD strain (JB631) was constructed and the ydcM/lepA ratio was determined and found to be very similar to that of strains JB137, JB134 and JB335 (Fig 2B). Thus, despite R-loop formation at this site, deleting it had no effect on the Ter peak height. Together, these results did not reveal the presence of strong oriK sites at the top of the Ter peak that could be responsible for over-replication in this region.

Deleting tus generated a flattened profile in MFA with no Ter peaks, improved growth and significantly corrected the chromosome segregation defect of a topA topB null mutant Another approach was used to investigate for the presence of strong oriKs in the Ter region. MFA profiles show that counterclockwise and clockwise moving replication forks within the Ter region are blocked by the TerA/Tus and TerB/Tus barriers, respectively (e.g. JB137, Fig 2A). Thus, if strong oriKs are present in the Ter region, deleting tus should lead to a clear reduction in Ter peak height but the peak should still be visible, whereas if no active oriKs are present a flattened profile should be generated. Fig 3A shows that the MFA profile of a topA topB tus strain (JB260, no spc) is clearly flattened with no peak at the genomic position identified in strain JB137 (Fig 2A). The absence of significantly active oriKs in the Ter region is further confirmed by the profile of spectinomycin-treated cells (JB260, spc), where replication from oriC was fully inhibited and RLDR was still active (Fig 3A). That RLDR was active in JB260 cells is shown below. A similar result, a flattened MFA profile in the Ter region upon tus deletion, was the strongest evidence for the absence of oriKs in the Ter region of rnhA null cells [50]. The JB260 MFA profiles (Fig 3A) also show the absence of significantly active oriKs outside the Ter region. Thus, these results show that: 1- RLDR could be the result of weak and widely distributed oriKs across the genome that are stochastically activated in the cell population as previously suggested [60] and 2- The presence of the prominent Ter peak in topA topB null cells could reflect over-replication associated with the process of replication fork fusion at Ter/Tus barriers. This is supported by the data in the next section. In this context, RLDR may contribute to over-replication by increasing the number of forks trapped at Ter/Tus barriers for a long period of time. PPT PowerPoint slide

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TIFF original image Download: Fig 3. MFA shows that deleting tus and inverting the TerB/Tus barrier, respectively, eliminates and re-localizes the high peak in topA topB null cells. MFA by NGS of genomic DNA extracted from (A) JB260 (ΔtopB ΔtusB topA20::Tn10 gyrB(Ts)) and (B) JB136 (ΔtopB ΔyncE topA20::Tn10 gyrB(Ts) IN(1.52–1.84)) cells grown at 30°C to log phase and treated (spc) or not treated (no spc) with spectinomycin for two hours. The inverted TerB barrier in JB136 is shown in blue (TerB is located within the IN(1.52–1.84) inversion in JB136 (inv)). See legend to Fig 2 for more details. https://doi.org/10.1371/journal.pgen.1010754.g003 Growth, cellular morphology, and nucleoid shape parameters of JB260 (topA topB tus) were compared with those of JB137 (topA topB). Fig 4A shows that JB260 cells grew significantly better than JB137 cells. Microscopy analysis shows that JB137 cells were generally much longer than JB260 cells and that their DNA was not evenly distributed (Fig 4B for pictures and 4C for quantitative analysis). Thus, these results strongly suggest that the growth and chromosome segregation defects of topA topB null cells are largely related to over-replication in the Ter region. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Deleting tus or inverting the TerB barrier slightly improve growth and significantly correct the filamentation and chromosome segregation defects of topA topB null cells. Cells of RFM443 (wild-type), JB137 (ΔtopB topA20::Tn10 gyrB(Ts)), JB260 (ΔtopB ΔtusB topA20::Tn10 gyrB(Ts)), JB136 (ΔtopB ΔyncE topA20::Tn10 gyrB(Ts) IN(1.52–1.84)), and JB206 (topA20::Tn10 gyrB(Ts)) strains were grown overnight at 37°C on LB plates and diluted in fresh liquid LB medium for growth curve at 30°C (A) or for growth at 30°C to an OD 600 of 0.8 for microscopy (B), as described in Material and Methods. Representative merged images of phase contrast and fluorescence pictures of SYTO-40-stained cells are shown in (B). In (C), cells for each strain (total number in parentheses) were examined in merged images to calculate the percentage of cells in each category. https://doi.org/10.1371/journal.pgen.1010754.g004

Analysis of the MFA profile of a topA topB null strain carrying a DNA inversion including TerB supports the hypothesis of over-replication being triggered by fork fusion events at Ter/Tus barriers While we were constructing topA topB yncE strains, we isolated a clone that had a much lower ydcM/lepA ratio than other topA topB yncE clones. This clone, strain JB136, carries an inversion that changes the position of the over-replicated region normally located in Ter (see Materials and Methods). The MFA profile revealed that the inverted region in JB136 includes TerB, so that this site now becomes the left boundary of the over-replicated region whereas TerG, a weaker barrier [38], is now the right boundary (Fig 3B, JB136). The high level of DNA replication on the right side of TerG illustrates both the weak activity of this site as a barrier and the high level of replication originating from the TerB-TerG interval. Strikingly, the peak between TerB and TerG in JB136 was as high as that between TerA and TerB in JB137 (compare JB137, Fig 2A, with JB136, Fig 3B), despite the lack of oriKs in this area as shown in the absence of a Ter/Tus barriers (Fig 3A, JB260, topA topB tus, no prominent peaks between TerB and TerG; no significant peaks also in this area in JB137 (Fig 2A)). This mimics the situation with JB137, where the absence of Tus (JB260) eliminated the prominent peak between TerA and TerB. It can also be seen that the copy number is still very high after TerG for more than 1 Mbp toward oriC and that in fact the highest copy number is not located at oriC (Fig 3B, spc). This is consistent with the presence of replication forks moving toward this oriC region. This is better illustrated by the MFA profile of JB136 relative to JB137 (S2 Fig). Altogether, our results are consistent with a model in which over-replication is mostly triggered by replication forks trapped at Ter/Tus barriers (mainly TerB here). Growth, cellular morphology, and nucleoid shape parameters of JB136 (topA topB ynce IN(1.52–1.84)) were compared with those of JB137 (topA topB). JB136 grew slightly better than JB137 (Fig 4A) whereas JB137 cells were generally much longer than JB136 cells and their DNA was not evenly distributed (Fig 4B for pictures and 4C for quantitative analysis). These results show that the chromosome segregation defect of topA topB null cells is mostly due to over-replication specifically in the Ter region.

The absence of topo III in topA null cells dramatically increase the Ter peak height but not R-loop formation and RLDR Although MFA data from our lab have previously shown that a Ter peak was present in single topA null strains, it was much less prominent than the Ter peak found in topA topB null strains [14]. Here, this is illustrated by the ydcM/lepA ratio showing that the Ter peak of the topA null strain is lower by 2.5 times than that of the topA topB null strain (Fig 5A, compare RFM443 (wild type), JB206 (topA) and JB137 (topA topB)), even though topA topB null cells grew slightly better than topA null cells at 30°C (Fig 4A). As previously demonstrated by MFA (), Fig 5A also shows that overproducing RNase HI significantly reduced the Ter peak height in topA topB null cells (topA topB cells overproducing (JB511) or not (JB512) RNase HI). This effect of RNase HI overproduction was shown to be mediated by the inhibition of R-loop formation that in turn inhibited RLDR [14]. Thus, we tested if the very strong increase in Ter peak height could be due to a dramatic increase in R-loop formation and RLDR when topB was also absent in topA null cells. A dot-blot experiment with S9.6 antibodies recognizing DNA:RNA hybrids was performed as previously to show R-loop formation in topA topB null cells [14]. As a control, the accumulation of R-loops in a rnhA null strain (MM84) but not in a wild-type strain (RM443) is shown in Fig 5B (Top left, RFM443 and right, MM84). Fig 5B also shows that R-loops accumulated only at a slightly higher level in topA topB null compared with topA null cells (bottom left, JB137 and right, JB206). PPT PowerPoint slide

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TIFF original image Download: Fig 5. The absence of topB in topA null cells dramatically increases the ydcM/lepA ratios (Ter peak height) but not R-loop formation. (A) ydcM/lepA ratio determined by qPCR of genomic DNA extracted from RFM443 (wild-type), JB206 (topA20::Tn10 gyrB(Ts)), JB137 (ΔtopB topA20::Tn10 gyrB(Ts)), JB511 (ΔtopB topA20::Tn10 gyrB(Ts) pSK760), and JB512 (ΔtopB topA20::Tn10 gyrB(Ts) pSK762c) cells grown at 30°C to log phase as described in Materials and Methods. pSK760 but not pSK762c carries the wild-type rnhA gene to overproduce RNase HI. (B) Dot-blot with S9.6 antibodies of genomic DNA from RFM443 (wild-type), MM84 (rnhA::cam), JB137 (ΔtopB topA20::Tn10 gyrB(Ts)), and JB206 (topA20::Tn10 gyrB(Ts)) cells grown at 30°C. +RNase HI indicates that the genomic DNA was treated with RNase HI. https://doi.org/10.1371/journal.pgen.1010754.g005 A protocol to detect RLDR (cSDR) was developed in our lab [27,61]. This procedure allowed the detection of such RNase HI-sensitive DnaA-independent replication in both topA and topA topB null mutants. In this protocol, log phase cells are first treated with spectinomycin for two hours to inhibit the initiation of replication from oriC and to allow the already initiated DnaA-dependent replication to be terminated. EdU, a thymidine analog, is then added to the cells to detect RLDR. The cells are fixed after one hour, and the Alexa Fluor 488 dye is conjugated to EdU via the click chemistry. EdU incorporation is detected by flow cytometry. Fig 6, top, shows the results for the wild-type strain (RFM443). The left panel is the no-EdU control where the peak represents non-specific binding of the dye to the cells (this peak is on the left side of the vertical line in the diagram). A similar peak was seen for all the strains when EdU was not added to the cells, and this control is therefore not shown for the other strains. The middle panel represents cells incorporating EdU (no spectinomycin, cells replicating their DNA; the peak of replicating cells is on the right side of the vertical line in the diagram). The right panel shows the cells treated with spectinomycin for two hours before the addition of EdU. In the case of RFM443, the result demonstrates the absence of RLDR in wild-type cells. The numbers at the top right of the panels represent the percentage of cells replicating their DNA. PPT PowerPoint slide

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TIFF original image Download: Fig 6. The absence of topB in topA null cells do not increase the level of RLDR. Flow cytometry to detect RLDR in RFM443 (wild-type), MM84 (rnhA::cam), JB206 (topA20::Tn10 gyrB(Ts)), and JB137 (ΔtopB topA20::Tn10 gyrB(Ts)) cells grown at 30°C as described in Material and Methods. The left panel is a no EdU control showing the position of the peak for cells not incorporating EdU (non-specific binding of the Alexa Fluor 448 dye). The middle panels are EdU but no spc, showing growing cells replicating DNA. The right panels (spc) are cells treated with spectinomycin for two hours before adding EdU to reveal RLDR. Numbers at the top right of the panels represent the percentage of cells incorporating EdU (replicating DNA). https://doi.org/10.1371/journal.pgen.1010754.g006 RLDR was readily detected in the rnhA null (MM84), topA null (JB206), and topA topB null (JB137) strains (Fig 6, spc, right panels). Importantly, levels of RLDR were very similar in topA null (JB206 spc, right panel, 74.0% of cells replicating their DNA) and topA topB null cells (JB137 spc, right panel, 74.7% of cells replicating their DNA). RLDR was also detected in topA topB tus (JB260) and topA topB yncE IN(1.52–1.84) (JB136) cells, as expected (S3 Fig). Thus, in agreement with the results of dot-blot experiments with S9.6, RLDR levels were found to be similar in topA (JB206) and topA topB null cells (JB137) and, therefore, could not explain the 2 to 3 times higher Ter peak in topA topB null cells compared with topA null cells. Thus, this increase in Ter peak height is most likely related to the function of topo III in decatenation during replication. This is supported by the data presented below.

A strain carrying a naturally acquired compensatory gyrase mutation reduces RLDR and Ter over-replication in a topA topB null mutant The effect of deleting topB on the topA null strain DM800 (W3110 background) carrying a naturally occurring compensatory gyrB mutation (gyrB225) was also studied. [9,10,55]. Here, a topB null derivative of DM800 was constructed (strain JB305) and its growth was found to be only slightly improved by overproducing RNase HI (S7A Fig, compare JB305, JB354 (JB305/pSK760) and JB356 (JB305/pSK762c)). JB305 grew slightly better than JB137 although more slowly than JB303 (JB305, S7A Fig, JB137, Fig 4A and JB303, S5A Fig). Deleting topB in DM800 significantly stimulated transcription-induced negative supercoiling. Indeed, whereas no hyper-negatively supercoiled pACYC184Δtet5’ could be detected in DM800, such topoisomers were readily detected when topB was deleted (S7B Fig, compare lanes 1 and 2, DM800 with lanes 3 and 4, JB305, 37°C and 30°C; note that the gyrB mutation of DM800 is not thermo-sensitive). The stimulatory effect of the absence of topB in a topA null strain is also observed in strains with the gyrB(Ts) background at 30°C (S8 Fig. two-dimensional chloroquine gel, compare JB206 (topA) with JB137 (topA topB)). This suggests, as already proposed [14], that topo III can act as a backup for topo I, at least when topo I is absent. An alternative explanation is that when topo III is absent, more topo IV enzymes are required for decatenation during replication and therefore less are available to relax hyper-negatively supercoiled DNA. RLDR was also detected in DM800 and JB305, although at a lower level than in the other topA topB null strains JB137 and JB303, consistent with the presence of the gyrB225 allele that is also expressed at 30°C (S7C Fig, see JB354 (JB305/pSK760) and JB356 (JB305/pSK762c), and compare the low level of cSDR replication in DM800 with the higher one in topA null strains JB206 (Fig 6) and VS111 (S6 Fig)). The lower level of RLDR also led to a lower level of Ter over-replication compared with strain JB137 (S9 Fig. JB305, MFA profile–and + spc, compared with JB137, Fig 2A). S9 Fig also shows that a parC parE amplification was not detected in strain JB305. Thus, a naturally occurring gyrase mutation reduces enough RLDR to prevent the critical overloading of the decatenation capacity of topo IV in the absence of type IA topos.

The rpoB*35 mutation making RNAP backtracking-resistant, reduces RLDR and Ter peak over-replication in a topA topB null mutant Results from a previous study from our lab had suggested that RNAP mutations can affect R-loop formation in topA null mutants [68]. The rpoB108 (rpoB: β subunit of RNAP) mutation was isolated, as a mutation conferring a stringent-like RNAP phenotype [68,69]. Stringent-like RNAPs compensate for the lack of ppGpp, the signalling molecule of the bacterial stringent response [70]. A gyrB(Ts) topA null mutant carrying this rpoB108 mutation was shown to require less RNase HI activity to grow at 28°C than a gyrB(Ts) topA null mutant carrying the wild-type rpoB allele, which suggested that this mutation rendered RNAPs less prone to R-loop formation [68]. Supporting this hypothesis was the observation that the rpoB108 mutation reduced R-loop-dependent hypernegative supercoiling of a plasmid [68]. In another study, the best-studied mutation conferring a stringent-like phenotype, rpoB*35, was shown to reduce transcription-replication conflicts [71] by reducing RNAP backtracking [72]. Furthermore, this rpoB*35 mutation, by reducing RNAP backtracking, inhibited R-loop formation associated with this process [72]. More recently, the rpoB*35 mutation was shown to allow topA null mutants to survive, although fast-growing colonies appeared on plates, suggesting the accumulation of parC parE amplifications [60]. Here, effects of the rpoB*35 mutation on growth, cell morphology, RLDR and Ter over-replication in a topA topB null strain have been studied. Fig 10A shows that the presence of the rpoB*35 mutation did not affect the growth of topA topB null cells (JB137 (topA topB) vs. JB639 (topA topB rpoB*35)) and, importantly, unlike the case for topA topB null cells, RNase HI overproduction did not stimulate the growth of topA topB rpoB*35 cells (compare topA topB rpoB*35, JB639 (no plasmid), JB656 (pSK760) and JB657 (pSK762c)). Thus, similar to the situation described for the rpoB108 mutation [68], these results suggested that the rpoB*35 mutation made the topA topB null cells less prone to the formation of toxic R-loops. This is supported by the results showing that rpoB*35 reduced both RLDR (Fig 10B, JB639, the + spc peak is clearly lower than the -spc peak, whereas both -spc and + spc peaks are similar for JB137, Fig 6) and the Ter peak height (Fig 10C, JB639, ydcM/lepA ratio of 1.2 compared with 2.8 for JB137, Fig 2B) in topA topB null cells. Fig 10C also shows that JB639 carried a parC parE amplification (JB639, qseC/lepA ratio of 3.5). The rpoB*35 mutation significantly corrected the filamentation and chromosome segregation defects of topA topB null cells (S10A Fig, qualitative and S10B Fig, quantitative results). Thus, these results suggested that RNAP backtracking is, at least partly, responsible for the formation of toxic R-loops leading to RLDR in topA topB null mutants. PPT PowerPoint slide

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TIFF original image Download: Fig 10. The rpoB*35 mutation inhibiting RNAP backtracking suppresses the positive effect of RNase HI overproduction on growth and reduces both the ydcM/lepA ratio and RLDR in topA topB null cells. (A) Cells of JB137 (ΔtopB topA20::Tn10 gyrB(Ts)), JB639 (ΔtopB topA20::Tn10 gyrB(Ts) rpo*35) JB656 (JB639 pSK760) and JB657 (JB639 pSK762c) strains were grown overnight at 37°C on LB plates and diluted in fresh liquid LB medium for growth curve at 30°C as described in Material and Methods. (B) Flow cytometry to detect RLDR in JB639 (ΔtopB topA20::Tn10 gyrB(Ts) rpo*35) cells grown at 30°C as described in Materials and Methods. See the legend of Fig 6 for more details. (C) ydcM/lepA and qseC/lepA ratios determined by qPCR of genomic DNA extracted from JB639 (ΔtopB topA20::Tn10 gyrB(Ts) rpo*35) cells grown at 30°C to log phase as described in Materials and Methods. https://doi.org/10.1371/journal.pgen.1010754.g010

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