Broken replication forks trigger heritable DNA breaks in the terminus of a circular chromosome

Abstract

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.

Fig 1

(A) Circular map of the E. coli chromosome: oriC, dif and terD to terB sites are indicated. Numbers refer to the chromosome coordinates (in kb) of MG1655. (B) Linear map of the terminus region: chromosome coordinates are shown increasing from left to right, as in the marker frequency panels (see Figure 1C for example), therefore in the opposite direction to the circular map. In addition to dif and ter sites, the positions of the parS_pMT1 sites used for microscopy experiments are indicated. (C) MFA analysis of terminus DNA loss in the recB mutant: sequence read frequencies of exponential phase cells normalized to the total number of reads were calculated for each strain. Ratios of normalized reads in isogenic wild-type and recB mutant are plotted against chromosomal coordinates (in kb). The profile ratio of the terminus region is enlarged and the profile of the corresponding entire chromosomes is shown in inset. Original normalized profiles used to calculate ratios are shown in S1 Fig. The position of dif is indicated by a red arrow. The ter sites that arrest clockwise forks (terC, terB, green arrow) and counter-clockwise forks (terA, terD, blue arrow) are shown. (D) Schematic representation of focus loss in the recB mutant: Time-lapse microscopy experiments showed that loss of a focus in the recB mutant occurs concomitantly with cell division in one of two daughter cells, and that the cell that keeps the focus then generates a focus-less cell at each generation. The percentage of initial events was calculated as the percentage of cell divisions that generate a focus-less cell, not counting the following generations. In this schematic representation, two initial events occurred (generations #2 and #7) out of 9 generations, and focus loss at generation #2 is heritable. Panels shown in this figure were previously published in [19] and are reproduced here to introduce the phenomenon.

Fig 2. Model for terminus DNA loss in the E. coli recB mutant by formation of a σ-replicating chromosome.

A) In the first step, one chromosome arm is broken at a replication fork. In the example shown, this random initial DSB occurs on the clockwise replication fork, but the reaction is entirely symmetrical and breakage of the other replication fork can also form a σ-replicating chromosome with a tail ending at this first DSB random position. In a wild-type strain the broken chromosome arm is repaired by RecBCD- RecA-mediated homologous recombination (not drawn). In a recB mutant the DNA end is slowly degraded by the combined action of helicases and ssDNA exonucleases. In the example shown, the leading strand template is broken (or was interrupted prior to arrival of the replication fork), and the parental strand (black line) is linked to the lagging strand at the fork (green dashed line) by gap filling and ligation. The position of the ydeV::parS_pMT1 focus next to dif is indicated by a yellow star. B) The intact replication fork progresses toward the terminus while the broken chromosome arm, which carries a replication origin, segregates to the other cell half and is separated from the intact homologous sequence by septum formation. The ydeV::parS_pMT1 locus next to dif is duplicated. (C) At cell division, the linear arm in the terminus region is broken during cell division; in the presence of FtsK the septum closes on the KOPS convergence point, dif. Note that since the induction of the SOS response by dsDNA ends requires RecBCD, division is not prevented by the SOS-induced SfiA protein in a recB mutant. Septum closure is concomitant with the disappearance of the ydeV::parS_pMT1 focus from one daughter cell. The two dsDNA ends created by septum closure are slowly degraded, generating the first focus-less cell that contains a partial chromosome. The cell that shows a focus carries a circular sigma-replicating chromosome with a shortened tail, and an intact fork from the first replication round, which is slowed down by ter sites. D) After cell division, a new replication round is initiated. E) The first counter-clockwise replication fork and the new clockwise fork merge. The strands made by copying the intact circular strand (dashed blue and green lines, copies of the blue line) are linked to produce the circular part of a σ-replicating chromosome. The strands made by copying the linear part (dashed and full red lines, copies of the black-green line) are linked to produce a tail containing an entire chromosome. The enlarged tail carries a replication origin, it segregates to the other half of the cell. F) Septum closure cleaves the tail DNA in the terminus region, producing a σ-replicating chromosome as in step C and the second focus-less, originally containing a nearly full linear chromosome in which the terminus DNA sequences are slowly degraded. G) The σ-replicating chromosome with a short tail originally interrupted at dif is replicated. More cycles of replication-breakage events (steps E-F-G) will generate a focus-less cell at each generation and reset the tail length on the sigma-replicating chromosome to the distance between the dif site and the position of the intact fork at each cell division. Blue and black thick lines, original chromosome strands; red and green thick lines, DNA synthesized at the first generation; black and red thin lines, DNA synthesized at the second generation; purple thick line, septum; full lines represent leading-strands and dashed lines lagging-strands, arrows indicate the 3’ DNA ends; the positions of origins (ori, blue small circles) and dif sites are indicated; the position of the ydeV::parS_pMT1 locus is shown with a yellow star.

Fig 3. Only fork breakage accounts for heritable terminus DNA loss.

A. In a recB mutant, a random DSB in the replicated region is not repaired (A), but both replication forks can progress (B), until they merge in the terminus region and produce one intact chromosome and one linear chromosome interrupted at the position of the initial DSB (C). The slowly degraded dsDNA ends are not at dif and form independently of cell division. Blue and black thick lines, original chromosome strands; red and green thick lines, DNA synthesized at the first generation; full lines represent leading-strands and large dashed lines lagging-strands, narrow dashed lines represent degraded DNA, arrows indicate the 3’ DNA ends; the position of origins (ori, blue small circles) and dif sites is indicated. B. In a recA mutant, degradation of linear DNA by RecBCD limits terminus DNA loss. (Step A) in the recA mutant the reaction also starts by replication fork breakage. Pathway B: (B1) the dsDNA end is bound by RecBCD which entirely degrades the linear part of the σ-replicating chromosome. (B2) this DNA degradation produces an intact circular chromosome, and no focus-less cell is formed. Pathway C: (C1) the dsDNA end is not degraded prior to segregation and the septum closes on the tail dif site. (C2) the terminus DNA is cleaved by septum closure. In the focus-containing cell, degradation by RecBCD of the short tail produces a circular chromosome and prevents heredity. In the focus-less daughter cell, the linear chromosome will ultimately be fully degraded by RecBCD to produce an anucleate cell.

Fig 4. Terminus DNA loss in recA mutants.

(A) Time-lapse analysis of focus loss in recA (left panel), recA recD (middle panel) and recA recB (right panel) mutants. Time-lapse experiments were carried out on M9 glucose agarose pads at 30°C with pictures taken every 10 min. Cells contain ydeV::parS_pMT1 and express the ParB_pMT1 protein from the gene inserted into the chromosome. The numbers in the lower left corner of the pictures indicate the frame number. For reasons of space limitations some frames are skipped. Cells that generate a focus-less cell during division are circled with a full white line. Most often two foci can be seen before division, which shows that focus loss results from the degradation of a DNA sequence that has been previously replicated. Cells that have lost the focus are circled with a dashed white line. These focus-less cells generally do not divide. In the recA mutant example (left), focus loss is transmitted for one generation (images number 1 and 13) and then the focus-carrying cell returns to normal divisions (images 33–47). In the recA recD mutant transmission is increased compared to the recA mutant, two examples are shown. The cell on the left generates a focus-less cell at each cell division for 3 generations (transmitted event, images number 3, 15, 22) before returning to a normal division (images 27–36). The cell on the right generates a focus-less cell (image 3) and then divides normally once (images 15–18, non-heritable event). At the next generation each focus-containing cell undergoes a new initial event (image 36); these late initial events were counted but not used to quantify heredity since the following generations were not visible. In the recA recB example (right), a focus-less cell is generated during 5 consecutive generations. Examples of focus-less cell production from a cropped bacterium, but for which all frames taken every 10 min are shown, can be seen in S1 Video (recB), S2 Video (recA), S3 Video (recA elongated cells) and S4 Video (recA recD). A schematic representation showing the frequency of initial and heritable events is shown below the time-lapse images. (B) MFA analysis of terminus DNA loss in the recA (left panel), recA recD (middle panel) and recA recB (right panel) mutants. Experiments are realized and plotted as in Fig 1C. Original MFA data are shown in S2 Fig.

Fig 5. Terminus DNA loss in recA sbcB sbcD, recB sbcB sbcD and in recB ruvAB mutants.

A and C left panel: time-lapse experiments. Examples of heritable focus loss are shown in recA sbcB sbcD and in ruvAB recB mutants. Time-lapse experiments were carried out as in Fig 4. The numbers in the upper left corner of the pictures indicate the frame numbers. The double white arrows indicate the presence of two foci before division, which shows that focus loss results from the degradation of a DNA sequence that has been previously replicated. The yellow stars show cells that have lost the focus following division. These focus-less cells generally do not divide while the sister cell that has kept the ydeV:: parS_pMT1 site keeps growing and generates a focus-less cell at each division. B and C right panel MFA analysis. Ratios of DNA sequence coverage in recB sbcB sbcD versus sbcB sbcD mutants (B), and of recB ruvAB versus ruvAB mutant (C left panel) are shown. Original MFA data are shown in S3 Fig.

Fig 6. A Focus-less cell can form from any of the two ends of a chromosome linearized 3 kb from dif.

(A) Schematic representation of the terminus region in a linear chromosome interrupted at position 1585. terD to terB, dif, the parS sites used for microscopy experiments, and the hipA hipB genes are shown. (B) Schematic representation of non-heritable focus loss on linear chromosomes and micrographs showing examples of focus loss during growth of recB cells in which the chromosome is linearized 3 kb from dif and carries either yddW:: parS_pMT1 (left panels) or ydeV:: parS_{pM T1} (right panels). Time-lapse experiments were carried out as in Fig 4A. Cells that generate a focus-less cell during division are circled with a full white line. Cells that have lost the focus are circled with a dashed white line. Loss of the yddW::parS_pMT1 focus occurring in image 5 (left panel) is not heritable, but focus-less cells divide because the hipA hipB genes are intact. Loss of the ydeV::parS_pMT1 focus occurring in image 17 (right panel) is not heritable, and focus-less cells do not divide because hipB is degraded. Another example of ydeV:: parS_pMT1 focus loss from a linear chromosomes is shown in S6 Video and the complete movie corresponding to the yddW:: parS_pMT1 images shown here is shown in S7 Video. C. Ratio of normalized sequence reads in RecB+ over recB mutant cells with a linear chromosome. Because hipAB is next to dif, cells that degrade this chromosome end do not multiply because they are blocked by the HipA toxin and become underrepresented in the population. Cells that degrade the other chromosome end multiply, which increases their relative amount in the population. Consequently, DNA loss in the population is amplified on the yddW::parS_pMT1 side and underestimated at the other end. Original normalized profiles used to calculate ratios are shown in S4 Fig. We observed that our linear strain carries a deletion of about 50 kb around positions 1400 to 1450, which was not observed previously and may be specific for our isolate.

Fig 7. Terminus DNA loss occurs in a matP recB mutant.

(A) and (B) Micrographs showing ydeV:: parS_pMT1 focus behaviour during growth of matP and matP recB cells. Arrows indicate segregating ydeV:: parS_pMT1 foci. Cells that generate a focus-less cell during division are circled with a full white line. They contain non-segregating ydeV:: parS_pMT1 foci and give rise to a focus-less cell (circled with a dashed white line) in a heritable way (image 13 and 28). (C) Ratios of normalized reads in isogenic matP recB mutants and matP RecB⁺, (D) Ratios of normalized reads in isogenic matP ftsK^ΔCTer recB mutants and matP ftsK^ΔCTer RecB⁺ cells. Ratios are plotted against chromosomal coordinates (in kb) and original normalized profiles used to calculate ratios are shown in S5 Fig.

Fig 8. Cephalexin treatment reveals a lack of terminus segregation.

A, B and C Micrographs show examples of highly intense, non-segregating ydeV:: parS_pMT1 foci (A and B, yellow arrows) or regions in the filaments that are devoid of ydeV:: parS_pMT1 focus (C, yellow arrows). D Percentage of cells with abnormal filaments in various mutants. In all strains except for the linear recB mutant, abnormal filaments contained focus-less regions associated with very intense, non-segregated foci (as shown in panel A and B). In the linear recB mutant, non-segregated highly intense foci were not observed, and abnormal filaments showed focus-less regions associated with well-segregated foci (as shown in panel C). Because of some variations from experiment to experiment, all results are shown, and we cite the two extreme values in the text. Blue square and blue line ydeV:: parS_pMT1 foci, pink triangles and pink lines yoaC:: parS_pMT1 foci.