It was recently reported that the recBC mutants of Escherichia coli, deficient for DNA double-strand break (DSB) repair, have a decreased copy number of their terminus region. We previously showed that this deficit resulted from DNA loss after post-replicative breakage of one of the two sister-chromosome termini at cell division. A viable cell and a dead cell devoid of terminus region were thus produced and, intriguingly, the reaction was transmitted to the following generations. Using genome marker frequency profiling and observation by microscopy of specific DNA loci within the terminus, we reveal here the origin of this phenomenon. We observed that terminus DNA loss was reduced in a recA mutant by the double-strand DNA degradation activity of RecBCD. The terminus-less cell produced at the first cell division was less prone to divide than the one produced at the next generation. DNA loss was not heritable if the chromosome was linearized in the terminus and occurred at chromosome termini that were unable to segregate after replication. We propose that in a recB mutant replication fork breakage results in the persistence of a linear DNA tail attached to a circular chromosome. Segregation of the linear and circular parts of this “σ-replicating chromosome” causes terminus DNA breakage during cell division. One daughter cell inherits a truncated linear chromosome and is not viable. The other inherits a circular chromosome attached to a linear tail ending in the chromosome terminus. Replication extends this tail, while degradation of its extremity results in terminus DNA loss. Repeated generation and segregation of new σ-replicating chromosomes explains the heritability of post-replicative breakage. Our results allow us to determine that in E. coli at each generation, 18% of cells are subject to replication fork breakage at dispersed, potentially random, chromosomal locations.
The bidirectional replication of the Escherichia coli circular chromosome starts at the replication origin oriC and ends when forks meet in the opposite region, the chromosome terminus. Replication forks are arrested in the terminus region by specific sites called ter where binding of the Tus protein blocks replication forks in an orientation-specific manner (reviewed in [1,2]). ter sites are oriented to form a replication fork trap, replication forks can enter the trap but their exit is delayed by pauses at several successive ter sites (Fig 1A and 1B). As chromosome segregation is concurrent with replication in bacteria, the origin and terminus regions are also the first and the last DNA sequences to be segregated during chromosome partitioning [3–5]. Following replication initiation, the two origins first remain associated at mid-cell for about 20 min and then move to the ¼ and ¾ positions of the cell. Then, the chromosome arms segregate from mid-cell to these positions as they are replicated. Finally, the terminus regions are also replicated at mid-cell and only separate shortly before cell division [3–5]. The chromosome terminus is organized in a large Ter macrodomain of about 780 kilobases (kb) by binding of the MatP protein to specific DNA motifs, the matS sites [6]. MatP also interacts with the septum protein ZapB, and thus maintains the Ter macrodomain at midcell during septum formation [7–9]. The terminus region is centred on a specific site called dif, the target of recombinases XerC and XerD for chromosome dimer resolution (reviewed in [10,11]). dif is positioned opposite oriC on the circular chromosome (Fig 1A), and is the inversion point of the GC strand skew. Specific motifs, KOPS (FtsK oriented polar sequences), which provide directionality of chromosome segregation, converge at the dif site (reviewed in [12]). They are recognized by the C-terminal domain of a septum-protein, the FtsK translocase which acts as an oriented DNA pump. KOPS motifs point from the origin of replication towards dif, allowing FtsK to bring newly replicated dif sites together at mid-cell and to remove DNA from the constricting septum [13,14]. As a result dif sites are the last region to be segregated away from mid-cell [5,15]. Recently a new phenomenon was described in the terminus region. Sequencing of the entire genome and analysis of DNA sequence coverage as a function of position on the chromosome (Marker Frequency Analysis, MFA) has revealed a deficit of sequences in the chromosome terminus region in the recB mutant [16–18] DNA double strand break (DSB) repair in E. coli is entirely dependent on homologous recombination, first steps of which are catalysed by RecBCD and RecA (reviewed in [20–22]). RecBCD is a heterotrimeric complex that binds to double-stranded DNA (dsDNA) ends. RecB and RecD are helicases, and RecB also acts as a nuclease. RecBCD degrades dsDNA ends until it encounters specific DNA motifs called chi sites, after which it continues to degrade the 5’ end. It then loads RecA on the protruding 3’ tail for homology search, strand invasion and strand exchange. The resulting Holiday junctions are resolved by RuvABC resolvase to generate recombination products. In the absence of RecA, DSBs lead to chromosome degradation because of the potent exonuclease activity of RecBCD. Indeed the complex was originally characterised as the major E. coli exonuclease, Exo V. recB and recC null mutants are deficient for DSB repair, but because the RecBC complex can still catalyze strand opening and RecA loading, recD mutants are Rec+. However, Exo V activity is abolished in all three null mutants, recB, recC and recD, even though the recD mutants still degrade linear DNA in vivo at 50% of the wild-type rate [23] Finally, RecBCD-dependent homologous recombination is coupled with replication restart, which allows chromosome replication to resume after the repair by homologous recombination of broken replication forks (reviewed in [24]). In a previous study we showed that the deficit of terminus DNA sequences observed in the chromosome of recB mutant cells, which we call terminus DNA loss (Fig 1C, S1 Fig), was independent of all known DNA processing events to take place in the terminus: replication fork merging, dimer resolution and decatenation of the two circular replicated chromosomes [19]. It also occurred in cells lacking FtsK-mediated chromosome segregation, but in an ftsK mutant, terminus DNA loss became less centred at dif, indicating a role for FtsK in the positioning of the peak of DNA loss around the site of convergence of KOPS sequences [19]. Our study led to the following key observations: (i) terminus DNA loss occurred during septum closure and required cell division, (ii) a first cell division generated one daughter cell that lacked the terminus sequence, and one that retained it (the initial event), (iii) the daughter cell that carried the terminus sequence generated again a non-proliferating terminus-less cell and a viable terminus-containing cell, at each following generation (heritable, transmitted events; [19]; Fig 1D). Furthermore, our analysis by RecA ChIP suggested that these terminus DSBs did not occur in wild-type cells, and were thus caused by the absence of RecBCD [19]. Here we have taken forward our previous study and used MFA and cell biology techniques to understand these mysterious observations. We propose and test a model in which, in a recB mutant, replication fork breakage triggers a terminus DSB during cell division in a heritable manner. Our results allow us to conclude that in wild-type, untreated E. coli cells, chromosome DSBs occur mainly at replication forks, and to determine the frequency of spontaneous replication fork breakage to be ~18% per cell per generation.
We studied terminus DNA loss by a combination of MFA and microscopy analyses. For microscopy, we used strains that constitutively express the yGFP-ParBpMT1 fusion protein from a chromosome-inserted gene and carry a parSpMT1 site at one of three different loci (Fig 1B). Binding of yGFP-ParBpMT1 to its cognate recognition site allows the visualization of each parS sequence as a fluorescent focus [25]. Three different strains were used, which carry ydeV: : parSpMT1 between dif and terC, 10 kb from each, or yoaC: : parSpMT1 about 300 kb away from dif on the left replichore, or ycdN: : parSpMT1 about 500 kb away from dif on the right replichore [19] (Fig 1B, S1 Table). All experiments were carried out in M9 glucose medium (called M9 henceforth). Exponentially growing wild-type cells showed one or two foci. Cells with two foci depended on whether the parSpMT1 site was replicated and segregated and therefore decreased with distance of the site from the origin [25] (S2 Table). In a recB mutant ~30% of cells showed no dif-proximal focus (ydeV: : parSpMT1), and ~7–8% showed no dif-distal focus (yoaC: : parSpMT1, ycdN: : parSpMT1) [19] (Table 1, S2 Table). Time-lapse microscopy experiments allowed the real time visualization of focus loss in recB mutant cells: ~18% of the divisions produced a focus-less cell and a daughter cell with a focus [19] (“% initial events” in Table 1; S1 Video) and focus loss was heritable in ~75% of the cases [19] (Fig 1D; “% transmitted” in Table 1; S1 Video; these inherited events are not counted in the 18% initial events). The molecular model depicted in Fig 2 explains these observations and has been tested in the present work. The model is as follows: a dsDNA end formed by breakage of one replication fork, at a dispersed and potentially random chromosomal location, results in a structure called a σ-replicating chromosome. This consists of an entire circular chromosome covalently linked to a linear partial chromosome arm by one intact replication fork (Fig 2, step A). The linear arm is repaired by homologous recombination in wild-type cells, but remains unrepaired in a recB mutant, in which σ-replicating chromosomes have been proposed to prevent cell growth [26,27]. We propose that in a recB mutant the linear and circular parts of this σ-replicating chromosome segregate to the two halves of the cell, while the intact replication fork progresses toward the terminus, and pauses at the ter sites (Fig 2, step B). However, the linear arm of the σ-replicating structure necessarily passes through mid-cell and is processed by FtsK, which precisely positions dif in the constricting septum (Fig 2, step C). The trapped DNA is broken during cell division, producing one daughter cell containing a linear, partial chromosome (focus-less cell) and the other one containing a σ-replicating chromosome with a shortened tail (Fig 2, step C). The DNA ends made during septum closure are located near dif and are slowly degraded by exonucleases. A second round of replication is initiated at oriC (Fig 2, step D) and the tail of the σ-replicating chromosome is enlarged by the entire newly replicated sequence when the intact replication fork of the σ-replicating chromosome merges with the fork of the second replication round (Fig 2, step E). This new σ-replicating chromosome contains a complete linear chromosome attached to the terminus of a circular chromosome. The circular and linear parts segregate to daughter cells, and the region around the dif site, maintained in the path of the septum by the FtsK translocase, is cleaved again during cell division (Fig 2 step F). This accounts for the efficient transmission of the phenomenon to the progeny in recBC mutants, as terminus breakage creates again a circular chromosome with a short tail and therefore the cycle of events can resume (Fig 2, step G). Importantly, we propose here that the initial DSB occurs at a replication fork, because a DSB elsewhere in the replicated region would leave both forks intact (Fig 3A). Replication would produce a circular chromosome with no scar and a linear chromosome interrupted at a random sequence, which cannot account for our observations of heritable terminus DNA loss during division and DNA degradation centred on dif. In a recA mutant, dsDNA ends are acted upon by RecBCD and linear DNA is very efficiently degraded. We predicted that both the first linear tail created by fork breakage and the second, smaller linear tail created by division-induced breakage should be degraded by RecBCD in recA cells, reducing initial events and transmission of the phenomenon, respectively (Fig 3B). We observed that the percentage of focus-less cells was three-fold lower in the recA mutant (9%) than in the recB mutant (~32%, Table 1, S2 Table). Time-lapse experiments showed that focus loss occurred in recA cells with some of the characteristics of recB cells: it occurred most frequently at the septum, always at the time of cell division and in one daughter cell only (Fig 4A left panel; complete movie is shown in S2 Video). However, the proportion of initial events in the recA mutant was 7% of total divisions, nearly three-fold less than in the recB mutant (17. 7%, Table 1, Fig 4A left panel). Furthermore, transmission of the phenomenon to progeny was less efficient in the recA than in the recB mutant, since (i) ~37% of events were transmitted to progeny instead of ~75% in recB cells, and (ii) the number of successive generations undergoing terminus DNA loss was reduced compared to the recB mutant: for example, among the events that could be followed for more than 3 generations, 19 out of 27 continued focus loss in the recB mutant versus only 2 out of 12 in the recA mutant, the other ones mostly returning to normal growth. Note that the percentage of heritable events decreased from 13. 3% of all divisions in the recB mutant (75% of 17. 7% of the divisions) to 2. 6% in the recA mutant (37% of 7% of the divisions). Furthermore, 5–10% of divisions in the recA mutant were preceded by cell elongation, and some elongated cells produced focus-less cells (S3 Video). This cell elongation could result from a partial degradation of the long DNA tail, which might prevent a correct DNA segregation and, in turn, block septum formation until the following replication round. In addition, in recA mutant cells we observed a similar percentage of cells lacking the dif-proximal ydeV: : parSpMT1 locus and the yoaC: : parSpMT1 locus further from dif (~9%; Table 1; S2 Table), and no terminus DNA loss could be detected by MFA ([17]; Fig 4B left panel, S2 Fig). The recA mutants are known to lose entire nucleoids, and ~10% loss of terminus corresponds to such recA mutant cells without chromosomes [28]. We propose that DNA degradation by RecBCD extends further around DSBs, degrading the entire chromosome in the 9% focus-less recA cells and thus preventing detection of DNA loss by MFA. To test whether the lower efficiency of focus loss in the recA mutant results from the DNA degradation activity of RecBCD in the absence of RecA (Fig 3B), we used a recA recB mutant. The percentage of focus-less cells was similar in recA recB and recB mutants for the dif proximal site ydeV: : parSpMT1 and for the distal sites yoaC: : parSpMT1 and ycdN: : parSpMT1 (Table 1, S2 Table). Furthermore, time-lapse experiments showed that focus loss occurred at the time of division, in one cell only, and was transmitted to progeny (Fig 4A right panel). The frequency of initial events (21%, Table 1, Fig 4A right panel) and the high rate of transmission to progeny (83. 7%) were similar in recA recB to the RecA+ recB strain. Furthermore, the MFA profiles were similar in recA recB and recB mutants (Fig 1C, Fig 4B right panel, S1 and S2 Figs). This result shows that in a recA single mutant the frequency of terminus DNA loss is reduced due to the presence of RecBCD. In a recA recD mutant, DSBs are not repaired because homologous recombination is inactivated by the recA mutation, and dsDNA ends are slowly degraded because the recD mutation inactivates the Exo V activity of the RecBCD complex (the RecB nuclease is not active in the RecBC complex lacking RecD, reviewed in [20–22]). recA recD mutant chromosomes were analysed by MFA (Fig 4B middle panel, S2 Fig). Terminus chromosome degradation covered a much larger region and was less steep than in recB cells, but was still centred on dif, the region of GC skew inversion. We propose that terminus DSBs occur in recA recD cells and that the very broad zone of DNA degradation around the terminus is due to the processive and potent helicase activity of RecBC, which in the absence of RecD produces ssDNA from dsDNA ends efficiently, and thus facilitates the action of ssDNA exonucleases [23,29]. Microscopy experiments confirmed DNA loss of a larger terminus region in the recA recD compared to recB mutant cells, since 27. 3% of them lacked the dif-proximal ydeV-parSpMT1 focus, 23% lacked the dif-distal yoaC-parSpMT1 focus and only 11% lacked the ycdN: : parSpMT1 locus, the furthest from dif (Table 1, S2 Table). Time-lapse microscopy analysis of ydeV-parSpMT1 foci in recA recD cells showed that focus loss occurred as in the recB mutant: most often at the septum, always at the time of cell division and in one daughter cell only, and it was transmitted to the progeny (Fig 4A middle panel, another example is shown in S4 Video). The frequency of initial events was 16. 1% and these events were transmitted to progeny in 65% of the cases, without cell elongation (Table 1, Fig 4A middle panel). We conclude that terminus DNA loss is limited in recA cells by the Exo V activity of RecBCD. Recently, terminus DNA loss was also observed in a recA sbcB sbcD mutant [30]. In this mutant RecBCD is present but does not degrade DNA efficiently because DNA degradation requires dsDNA ends to be made blunt by SbcB and SbcCD exonucleases [31,32]. In agreement with a lack of DNA degradation by RecBCD in the recA sbcB sbcD mutant, microscopy results in the recA sbcB sbcD mutant were similar to the recA recB mutant (Table 1, Fig 5A), while inactivation of only sbcB or sbcCD in the recA mutant had a partial effect (Table 1). Finally, our model predicts that heritable terminus DNA loss should occur at a low efficiency in a recB sbcB sbcD mutant, which lacks RecBCD but where DSBs are repaired by the RecFOR pathway of recombination (reviewed in Michel and Leach, 2012). Actually in this mutant initial events were decreased nearly two-fold (to around 10%, Table 1) and focus loss was less frequently transmitted to progeny (27,3% heritable events, Table 1). These results are in agreement with the repair of dsDNA ends by the RecFOR recombination pathway, even though MFA analysis suggested that recB sbcB sbcD mutants initiate unscheduled replication in the terminus, and an unexplained high level of focus-less cells in growing cultures suggested that additional phenomena occur in the terminus region of the recB sbcB sbcD mutant ([30]; Fig 5B; S3C and S3D Fig; Table 1). Altogether, these results demonstrate that both homologous recombination and RecBCD-mediated DNA degradation should be inactivated to observe heritable terminus DNA loss, as predicted from our model (Figs 2 and 3). To date, only one particular replication fork breakage event is specific for recB and recA recD mutants, and those breaks result from RuvABC-catalysed resolution of a Holliday junction made by replication fork reversal [22,33]. Replication fork reversal is a reaction that involves the annealing of leading- and lagging-strand ends at a blocked fork, resulting in a dsDNA end adjacent to a Holliday junction [22,33]. In recBC and in recA recD mutants, the dsDNA end is neither recombined nor degraded, and resolution of the Holliday junction by RuvABC produces fork breakage [22,33]. Fork breakage by RuvABC in a recB mutant is a hallmark of replication fork reversal, and we tested a putative role of RuvABC in the production of the DSBs that lead to terminus DNA loss. The inactivation of ruvAB did not reduce the percentage of focus-less cells in recB ruvAB (37%, Table 1) or in recA recB ruvAB cells (38%, Table 1). Focus loss in the recB ruvAB and recA recB ruvAB mutants occurred at the time and most often at the site of cell division, in one daughter cell, and was transmitted to progeny (Fig 5C). Focus loss was quantified by time-lapse experiments in recA recB ruvAB cells, where only recombination-independent Holliday junctions can form. The frequency of initial events was unchanged by RuvAB inactivation (about 21%), and transmission of focus loss to progeny was slightly lower than in the Ruv+ recA recB mutant but remained high (60%). Furthermore, DNA loss in the dif region was still observed by MFA in the recB ruvAB mutant (Fig 5C, S3A and S3B Fig). We conclude that RuvAB is not required for terminus DNA loss in the recB mutant, which implies that replication fork reversal is not the main source of fork breakage in this mutant. The model predicts that the focus-less cell generated by the first cell division carries a truncated linear chromosome lacking all sequences between the original random DSB and the terminus, therefore potentially lacks essential genes. In contrast, focus-less cells generated in the following generations, which are delimited by two DSB events in the terminus region, contain a complete linear chromosome. This prediction could be tested by comparing the ability to propagate of these two types of focus-less cells. For this experiment we had to use a hipA hipB deleted strain since this toxin-antitoxin locus is adjacent to dif and its degradation in ydeV-parSpMT1 focus-less cells prevents proliferation [34,19,35]. In a hipA recB mutant 30% of the first focus-less cells did not divide while all the second focus-less cells divided (<3% did not divide, Table 2). This indicates that 30% of the first focus-less cells lacked some essential proteins that were expressed by the second focus-less cells. This is in agreement with the proposal that the first focus-less cells originally carry a truncated linear chromosome and thus differ from the subsequent focus-less cells that are born with a full linear chromosome. According to the model presented in Fig 2, transmission of the phenomenon to progeny requires the production of a σ-replicating chromosome, in which a linear and a circular chromosome are attached by a replication fork in their terminus (Fig 2 Step E). Therefore transmission should be prevented by using cells in which the naturally circular E. coli chromosome has been converted to a linear chromosome, artificially interrupted in the dif region. We used a strain that carries the terminus sequence tos of the linear phage N15,3 kb from dif on the right replichore, and that expresses the N15 telomerase TelN, which processes the tos sequence (Fig 6A) [36]. This strain propagates with a linear chromosome, interrupted 3 kb from dif [36]. As a control for these experiments, we used an isogenic strain with a circular chromosome, which carries the tos site but lacks the gene encoding the TelN protein (S1 Table). Cells with linear chromosomes were studied by fluorescence microscopy, using ydeV: : parSpMT1 or gusC: : parSpMT1 markers on the left replichore (13 kb or 105 kb from the chromosome end, respectively), and yddW: : parSpMT1 or pspE: : parS pMT1 markers on the right replichore, (19 kb or 217 kb from the chromosome end, respectively) (Fig 6A, Table 3). It should be noted that the hipA hipB locus is adjacent to dif, therefore it will be degraded together with the ydeV: : parSpMT1 or gusC: : parSpMT1 markers, inhibiting growth of these focus-less cells. In contrast, because it is separated from the other chromosome arm by the tos site, it will remain intact in cells that lose the yddW: : parSpMT1 or pspE: : parSpMT1 markers, allowing the multiplication of the cells that lose these loci. The proportion of cells lacking the end-proximal ydeV: : parSpMT1 focus increased from 4. 8% in the RecB+ strain to 20. 7% in the recB mutant, while the proportion of cells lacking the end-distal gusC: : parSpMT1 focus reached 10. 5% in the recB mutant (Table 3). In contrast, the proportion of the cells devoid of the end-proximal yddW: : parSpMT1 focus increased from 4. 1% in RecB+ to nearly 60% in the recB mutant, while the proportion of cells lacking the end-distal pspE: : parSpMT1 focus reached 56% in the recB mutant. As expected, in control isogenic strains with a circular chromosome, the proportion of cells lacking the dif-proximal loci (ydeV: : parSpMT1 or yddW: : parSpMT1) was increased from about 1% in RecB+ to around 30% in the recB mutant, and was higher than the loss of a dif-distal locus (pspE: : parSpMT1,15% focus-less cells in a recB mutant, Table 3). The difference between right and left replichores was specific for linear chromosomes, suggesting that the proportion of focus-less cells could be largely influenced by the position of the hipA hipB locus. To precisely quantify terminus DNA loss, ydeV: : parSpMT1 and yddW: : parSpMT1 foci were analysed in recB by time-lapse microscopy experiments. Results in the control recB mutant that carries tos but harbours a circular chromosome owing to the absence of TelN protein were similar to those observed in MG1655, with a loss of ydeV: : parSpMT1 or yddW: : parSpMT1 foci occurring at the time of cell division, in one of the two daughter cells, and transmitted to progeny (S5 Video). We counted 15. 9% initial events for the yddW: : parSpMT1 locus and more than 80% of the events were transmitted to progeny (Table 4). In cells with a linear chromosome, a similar percentage of initial events was observed with the terminus-proximal markers on the left and right replichores (14–17%) but, importantly, the phenomenon was generally not transmitted to progeny, as only 11 to 19% of the events were heritable (Table 4, note that this level corresponds to the percentage of initial events and could therefore correspond to independent events occurring by chance after a first one). This result indicates that the transmission of focus loss to the progeny requires circularity of the chromosome. In addition, time-lapse experiments allowed us to observe that ydeV: : parSpMT1 focus-less cells did not multiply, as expected from the concomitant degradation of the hipA hipB locus (Fig 6B right panels, another example of ydeV: : parSpMT1 focus loss from a linear chromosome is shown in S6 Video). In contrast, cells that lose the yddW: : parSpMT1 locus on the right replichore could multiply for at least three generations (Fig 6B left panels, complete movie is shown in S7 Video). Therefore, the high level of yddW: : parSpMT1 and pspE: : parSpMT1 focus-less cells can be simply explained by the propagation of focus-less cells carrying an intact hipA hipB locus. Genomes of the RecB+ and recB mutant linear strains were analysed by MFA (Fig 6C, S4 Fig). A depletion of DNA sequences was observed on one chromosome arm, while nearly no DNA loss was observed on the chromosome arm carrying hipA hipB, possibly because the MFA technique is not sensitive enough to detect the weak level of recB-dependent DNA loss on this arm (16%, Table 3). Although the MFA profile was therefore not informative regarding terminus DNA loss, it was in full agreement with the microscopy results. We conclude from these experiments that focus-less cells, which reflect a lack of terminus DNA, could be observed at either of the two ends of a recB mutant chromosome linearized at position 1585 kb. The phenomenon shares some common features with terminus DNA loss observed in circular chromosomes (focus loss in one daughter cell, at the time of division), but, importantly, the capacity to lose terminus DNA in one daughter cell was not heritable. These results indicate that chromosome circularity, and thus DNA continuity of the terminus region is required for the heredity of the phenomenon, although it is not required for the formation of a first focus-less cell (initial events). According to our model, transmission of terminus DNA loss to progeny depends on the persistence of the short DNA tail formed at each generation by septum closure until the arrival of the following replication round (Fig 2 step D). In a recB mutant, DNA degradation is mediated by the action of helicases and exonucleases and is expected to be much slower than RecBCD-catalysed DNA degradation [37,38]. In a recA mutant, this short tail is the target of the potent RecBCD Exo V activity and should be efficiently degraded, which explains why only 37% of the initial events, instead of 80% in the recA recB mutant, were transmitted to progeny at least for one generation. The length of this tail is defined by the distance between the site of breakage (the dif region) and the position at the time of division of the intact replication fork that is slowed down by ter sites (Fig 2 step C). Therefore, the duration of replication blockage at ter is expected to control heredity of terminus DNA loss in a recA mutant. We measured terminus DNA loss in a tus recA mutant, in which replication forks do not arrest at ter. tus inactivation increased the percentage of initial events from 7% to 11%, and increased the percentage of heritable events in the recA mutant from 37% to 64%, similar to the recA recD level (Table 1, S2 Table). This result shows that in a recA mutant replication arrest at ter limits terminus DNA loss and particularly the transmission of terminus DNA loss to the progeny. The model presented in Fig 2 implies that the two terminus sequences remain covalently attached. In wild-type cells, this covalent attachment cannot be directly visualized, as the two newly-synthesized terminus regions are anyway co-localized at the septum position when MatP is present. In contrast to wild-type cells, in a matP mutant terminus sequences readily separate after replication [6,7]. We used a matP mutant to test the attachment of the newly synthesized terminus sequences in the recB mutant. As previously described, all matP cells exhibited an early segregation of the ydeV: : parSpMT1 loci to the ¼ and ¾ positions in the cell, owing to the lack of attachment of the terminus macrodomain to the septum ([6] arrows in Fig 7A). MFA and microscopy experiments showed that terminus DNA loss occurred in matP recB as in the recB single mutant (Table 1, Fig 7B and 7C, S6 Fig). In time-lapse experiments, focus loss occurred at the septum, at the time of division, in one of the two daughter cells, and in a heritable manner (Fig 7B). Measures of initial events and heredity showed that DNA loss was unaffected by matP inactivation (15. 5% initial events, 86. 4% heredity; Table 1). However, although in most recB matP cells ydeV-parSpMT1 foci segregated prematurely to the ¼ and ¾ positions (arrows in Fig 7B), in ~15–16% of cells foci remained together at the site of septum formation until division (cells circled with a full white line in Fig 7B). Interestingly, focus loss occurred specifically in those cells where the two replicated ydeV: : parSpMT1 foci remained nearby in the division plane, or, in other words, the lost focus was always one of the two foci that remained at the septum position after replication, in spite of the absence of MatP (Fig 7B, focus-less cells are circled with a dashed white line). The specific loss of one of the two non-segregated loci in the matP recB mutant supports the idea that the two replicated chromosomes are linked at a position close to the ydeV locus (Fig 2). FtsK also contributes to the positioning of the chromosome terminus at the septum via binding of its C-terminal domain to KOPS sequences and chromosome translocation [5,39]. Nevertheless, in the matP ftsKΔCter recB mutant, which lacks the two functions known to position the terminus at the septum, ~40% focus-less cells were observed (Table 1). The MFA experiment showed an enlarged degraded region confirming that FtsK is not required for terminus DNA loss. Furthermore, DNA degradation was no longer centred on dif and spanned the entire fork trap, delimited by oppositely-oriented ter sites (Fig 7D), confirming that FtsK translocation activity is responsible for the localization of the peak of DNA degradation around dif. Importantly, terminus DNA loss is observed in the absence of the functions that position the chromosome terminus at the septum, which supports the idea that the two terminus sequences are attached covalently. To confirm the post-replication attachment of two terminus regions in a MatP+ strain, we analysed chromosome segregation using cells where division is blocked by cephalexin, an inhibitor of the late septum protein FtsI [40]. As expected, cephalexin treatment caused the formation of elongated cells, and most wild-type cells showed regularly spaced ydeV: : parSpMT1 foci, while 15–25% showed non-segregated foci (Fig 8A and 8D). The proportion of cells with non-segregated ydeV: : parSpMT1 foci was similar in all recombination proficient cells: between 11% and 25% non-segregated ydeV: : parSpMT1 loci (dif proximal) and between <0. 5% and 6. 3% non-segregated yoaC: : parSpMT1 loci (300 kb away from dif) (Fig 8D, see wild-type, recD, sbcB sbcD, recB sbcB sbcD and the circular chromosome control cell). Septum assembly is essential for dimer resolution owing to the role of the FtsK C-terminal domain in XerCD activation [41,42], and about 15% of cells contain a chromosome dimer [43]. Consequently, the percentage of recombination proficient cells showing non-segregated dif-proximal loci can be accounted for by the lack of dimer resolution. In support of this idea, because dimers only form in circular chromosomes, nearly all cells harbouring a linear chromosome showed proper segregation of ydeV: : parSpMT1 loci upon cephalexin treatment (0. 7% non-segregated, Fig 8D). In a recA mutant, 10–17% cells showed non-segregated ydeV: : parSpMT1 foci. Since chromosome dimers do not form in the absence of homologous recombination (recA mutant), these 10–17% cells suffer a dimer-independent segregation defect (Fig 8D). The marker further from dif (yoaC: : parSpMT1) was less affected and showed only 4% non-segregated cells. Inactivation of recD, recB or sbcB sbcD in the recA mutant increased the proportion of cells showing non-segregated ydeV: : parSpMT1 foci to 17–29%, therefore, in recA mutants the lack of segregation of the ydeV: : parSpMT1 foci after cephalexin treatment (Fig 8D) is correlated with the frequency of initial loss events (Table 1). This result supports the idea that terminus DNA loss occurs in cells in which the two termini remain covalently linked after replication. Interestingly, in the recB and recC mutants the percentage of cells presenting a segregation defect was as high as 22–44% for the dif proximal locus and increased to 9–14% for the dif-distal locus (Fig 8B and 8D). Since dimer formation is half as frequent in the recB mutant as in wild-type cells [43], the proportion of cells in which ydeV: : parSpMT1 foci did not segregate independently of dimer formation could be as high as 15–30%, as in recA recD and recA sbcB sbcD mutants. This percentage correlates with the level of terminus DNA loss observed in dividing cells (nearly 20% of initial events). Note that in cephalexin-treated cells focus segregation was similar to wild-type in recB sbcB sbcD (Fig 8D), although this mutant showed an intermediate level of initial events between wild-type and recB mutant, (10%, Table 1). To account for this observation, we propose that dsDNA end repair is slower when catalysed by RecFOR and RecA (recB sbcB sbcD cells) than when catalysed by RecBCD and RecA (wild-type). Consequently, in recB sbcB sbcD cells that do not divide (cephalexin treated), initial DSBs are repaired, although slowly, which allows segregation of sister chromosomes, while in dividing cells σ-replicating chromosomes are not always repaired prior to division and are sometimes cleaved. Finally, as expected from its high level of initial events, the recB mutant with a linear chromosome showed a high level of cells with an abnormal pattern of ydeV: : parSpMT1 foci after cephalexin treatment (24–40%). However, in the linear chromosome recB mutant the abnormal cephalexin-induced filaments presented a deficit of ydeV: : parSpMT1 foci (Fig 8C) instead of non-segregated foci, as observed in recB cells and in other mutants with a circular chromosome (Fig 8B). As described below this is expected from the random breakage of one replication fork in a linear chromosome (S6 Fig, see Discussion). In conclusion, a defect in segregation of the two replicated dif regions is observed in cells that lack homologous recombination and Exo V mediated DNA degradation both in the presence (in a matP mutant) and the absence (in cephalexin-treated cells) of cell division. This finding supports the idea that terminus DNA loss results from septum closure on non-separated chromosome termini.
Initial events rely on the persistence of a σ-replicating chromosome tail after fork breakage, which can lead to a focus-less cell only if the linear tail is neither degraded nor recombined, and segregates to the future daughter cell (Fig 2B and 2C). The observation that initial events are three-fold less frequent in recA than in a recA recB mutant suggests that in two thirds of cases the potent Exo V activity of RecBCD (variable but up to 800–900 bp per sec, [44,45]) catches up with the progressing fork (500–600 bp per sec, [46,47]) and fully degrades this first long tail, which prevents initial events (Fig 3B pathway B). In a recA mutant the frequency of both initial and secondary events is increased by tus inactivation. The increase of initial events could be explained by two ways. Firstly, complete DNA degradation of the first tail is expected to be delayed by the progression of the active replication fork across the terminus. Secondly, in a subpopulation of cells, the progression of one of the two intact replication forks beyond the terminus, in the direction opposite to the main transcription direction, might increase replication fork blockage, as previously proposed, and in turn replication fork breakage and σ-replicating chromosome formation [48,49]. Increased heredity in the recA tus compared to the recA mutant supports the idea that heredity relies on the persistence of the truncated tail after terminus DNA breakage, hence on the length of this tail (Fig 2D–2F). Growing cultures of recA mutants were reported to contain 5 to 10% anucleate cells (see for example [32,50]), which corresponds to the percentage of focus-less cells observed in this work. Interestingly, in the recA mutant we did not observed loss of parSpMT1 foci at any time other than cell division. This observation suggests that most anucleate cells in MM cultures of a recA mutant result from the degradation of a linear chromosome formed by two successive DSBs: one at a random position during replication and one close to dif during septum closure (Fig 3B pathway C). The formation of a focus-less cell is not transmitted to progeny when the chromosome is linearized by tos/TelN, in agreement with the idea that heredity requires circularity of the chromosome for the merging of the intact replication fork with the following replication round (Fig 2). A model showing the events expected to occur in the recB mutant harbouring a linear chromosome, according to the model shown in Fig 2, is presented in S6 Fig. In the recB mutant with a linear chromosome, accidental breakage of one replication fork, while the other replication fork progresses to the chromosome end, leads to a linear head-to-head dimer composed of one entire chromosome and one truncated chromosome, linked by the telomerase TelN recognition site (S6 Fig, 3 first steps). The two halves of this dimer segregate to the two future daughter cells, with the TelN recognition site at mid-cell. TelN action at this site produces an intact linear chromosome, which segregates to form the focus-carrying cell, and a truncated chromosome (focus-less cell). Cells that harbour a truncated chromosome lacking the ydeV site do not multiply while those that lack the yddW locus multiply. Note that the reaction starts by fork breakage as on a circular chromosome, but the missing terminus, which fails to be copied by the broken replication fork, is not copied by the other fork (and then degraded), since the chromosome is linear (S6 Fig, progression of the intact fork to the end). Accordingly, in time-lapse experiments we did not observe a duplication of the ydeV: : parSpMT1 or yddW: : parSpMT1 focus prior to focus loss (Fig 6), and after cephalexin treatment abnormal elongated cells showed regions devoid of focus (Fig 8). Linearization in the terminus by TelN separates the intact from the truncated linear chromosomes after replication completion (S6 Fig, last step), and no DSB occurs during cell division. Our results account for the long-standing observation of three types of cells in a recB mutant culture: non-dividing cells (our focus-less cells), residually dividing cells (the cells that produce a focus-less cell), and normally dividing cells [51], Furthermore, the viability of recB cells is lower than that of recA mutant cells although, in addition to DSB repair, the latter also lack single-strand gap recombinational repair and induction of all DNA repair genes under the control of the SOS response [51–53]. It was proposed that the tail of a σ-replicating chromosome is a lethal form of damage in a recBC mutant, and that σ-replicating chromosomes are less deleterious in a recA mutant where the linear tail can be degraded by RecBCD [26,27,52]. Our study strongly supports the idea that σ-replicating chromosomes are the major cause of the low viability of the recB mutant but they do not simply cause lethality. Instead, one cell remains alive while most of the tail is segregated and cleaved off into a doomed daughter cell at each generation. Several kinds of replication impairments render RecBC, and sometimes also RecA, essential for viability [54,55]. The reverse assumption, that the viability defect of recBC and recA mutants directly reflects a correspondingly high level of spontaneous replication impairment, was often postulated. However, in contrast with this assumption, flow cytometry and MFA analyses showed that chromosome replication proceeds with a rate similar to wild-type in recB and recA mutants [19,56]. Replication fork blockage or breakage was not observed, although it should have been detected if it were responsible for the low viability of these mutants. Our model provides an explanation for this paradox. Our data allow us to determine for the first time that the level of spontaneous replication fork breakage is ~18% per cell per generation (9% per fork), which is too low to be directly detected in population studies. Finally, our findings raise future questions to be addressed: how does spontaneous replication fork breakage occur, and how are terminus DSBs catalysed? We have previously shown that the periplasmic endonuclease Endo 1 is not involved [19] and no nuclease has been reported to be specifically associated with the septum.
Strains are described in S1 Table. Most strains were constructed by P1 transduction. New mutations were constructed as described in [57], using DY330 [58]. Oligonucleotides used for constructions and mutation checking are shown in S3 Table. recA and recB mutations were checked by measuring UV sensitivity. recD mutations were checked by comparing the plating efficiencies of wild-type T4 and T4gpIIam phages (the unprotected T4gpIIam only multiplies on recBC and recD mutants [59]). sbcCD mutations were checked by comparing the plating efficiencies of wild-type λ a λ carrying a long palindrome (the λDRL154 phage that carries a long palindrome only multiplies on sbcCD mutants, [60]). In the course of this work, we fortuitously discovered that our microscopy strains are Phi80 lysogens. In contrast with the reported effects of Phi80 lysogeny in AB1157 [61,62], Phi80 lysogens in MG1655 are only very weakly UV sensitive (around 10% survival at 40 J/m2), do not affect T4 or λ phages plating, and do not show a recD or sbcCD mutant phenotype. These background differences presumably result from the high divergence of the AB1157 and MG1655 genomes. All strains used for MFA are Phi80-free and experiments with Phi80-free recA and recB mutants confirmed that the cryptic phage did not affect the microscopy results (S4 Table). MFA were performed and analysed as described in [19], with cells grown in M9 glucose at 37°C. The MFA data have been submitted to the ArrayExpress repository. The access number for these data is E-MTAB-6122. Microscopy experiments were performed and analysed as described in [19]. For snapshot analysis cells were grown in M9 glucose at 37°C. Time-lapse experiments were realized on M9 glucose at 30°C.
The Escherichia coli recBC mutant, deficient for DNA double-strand break (DSB) repair, shows a viability defect and a specific deficit in the level of chromosome terminus DNA sequences. We previously showed that this deficit results from heritable terminus DNA loss, owing to cell-division dependent DSBs in the chromosome terminus. Here, we used whole genome sequencing and microscopy to analyse the phenomenon. Our results allow us to conclude that in E. coli most spontaneous DSBs occur at replication forks, and that such breaks occur in 18% of cells at each generation. In a recBC mutant the linear chromosome arm made by replication fork breakage is neither repaired nor degraded. Thus it remains attached to the circular chromosome part, which triggers a DSB in the chromosome terminus during cell division in a heritable reaction. In wild-type cells, broken replication forks are repaired and these terminus DSBs do not occur. Our study reconciles the idea that replication fork impairment is a major source of chromosome breakage with the observation that most DSBs in a recBC mutant occur in the chromosome terminus during cell-division and reveals the links between these two phenomena.
chromosome structure and function
cell cycle and cell division
cell processes
dna replication
forms of dna
circular dna
dna
homologous recombination
chromosome biology
genetic loci
biochemistry
cell biology
nucleic acids
heredity
genetics
biology and life sciences
dna recombination
chromosomes
Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome