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A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

  • Esma Bentchikou,

    Affiliation Université Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Orsay, France

  • Pascale Servant,

    Affiliation Université Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Orsay, France

  • Geneviève Coste,

    Affiliation Université Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Orsay, France

  • Suzanne Sommer

    suzanne.sommer@igmors.u-psud.fr

    Affiliation Université Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V, Institut de Génétique et Microbiologie, Orsay, France

Abstract

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.

Author Summary

Deinococcus radiodurans bacterium is among the best-known organisms found to resist extremely high exposures to desiccation and ionizing radiation, both causing extensive DNA double-strand breaks. Because a single unrepaired DNA double-strand break is usually lethal, DNA double-strand breaks are considered as the most severe form of genomic damage. The extreme radioresistance of D. radiodurans is linked to its ability to reconstruct a functional genome from hundreds of chromosomal fragments. Genome reconstitution occurs through a two step process: (i) an extended synthesis-dependent strand-annealing process (ESDSA) that assembles genomic fragments in long linear intermediates that are then (ii) processed through recombination to generate circular chromosomes. Here, we demonstrate the essential role of key components of the D. radiodurans RecF pathway in ESDSA. We show that (i) inactivation of only one exonuclease (RecJ) results in cell lethality; (ii) cells devoid of RecF, RecO, or RecR display greatly impaired growth; (iii) RecF, RecO, or RecR proteins are essential for radioresistance through ESDSA; and (iv) UvrD helicase has an unexpected crucial function in DNA double-strand-break repair through ESDSA.

Introduction

The bacterium Deinococcus radiodurans is extremely resistant to treatments such as ionizing radiation and desiccation. This resistance can be correlated with the ability of D. radiodurans to reconstruct a functional genome from hundreds of radiation or dessication-induced chromosomal fragments, while the genomes of most organisms are irreversibly shattered under the same conditions. The rapid reconstitution of an intact genome is dependent on extended synthesis-dependent strand annealing (ESDSA) and recombination [1],[2]. It was proposed that, following severe DNA damage, the fragmented DNA end is recessed in a 5′–3′ direction, liberating single stranded 3′ overhangs which, through RecA- and RadA-mediated strand invasion, prime DNA synthesis on overlapping fragments [2]. DNA synthesis is initiated by Pol III and elongated by Pol I or by Pol III and the newly synthesized single-strands anneal to complementary single stranded extensions forming long double stranded DNA intermediates which are assembled into intact circular chromosomes by RecA-mediated homologous recombination [2]. Though the dependence of ESDSA on RecA, Pol I, and Pol III activities is well documented [1],[2], little is known about the cellular factors required for the first steps of this process (i.e. the formation of the single stranded 3′ overhangs which promote RecA/RadA - dependent strand invasion to prime DNA synthesis).

Three enzymatic activities are required for presynaptic processing of double stranded DNA ends in the model bacterium Escherichia coli: a helicase, a 5′-3′exonuclease, and a mediator function for efficient RecA filament formation onto ssDNA (see for reviews [3][5]). All these activities are carried out by the RecBCD complex (or its functional homolog AddAB) which is the major component for initiation of recombinational repair of DNA double-strand-breaks (DSB) in wild-type cells. However, if RecBCD is inactivated, an alternate pathway, the RecF pathway, promotes recombinational DSB repair [6][10] in cells containing mutations in sbcB (suppressor of recBC), which encodes the 3′-5′ exonuclease I, and in sbcC (or sbcD) [11]. This pathway comprises the 5′-3′ single-strand DNA exonuclease RecJ, the RecQ helicase and the RecF, RecO and RecR proteins that act together to promote loading of RecA onto single stranded DNA.

Whereas examination of the phylogenetic distribution of RecBCD and AddAB complexes revealed that one or the other complex is present in most sequenced bacteria, D. radiodurans is naturally devoid of these two complexes but does encode a RecD homologue [12]. RecD protein was shown to be present in the absence of RecBC not only in D. radiodurans, but also in firmicutes and Streptomyces [13]. The deinococcal RecD protein is expressed and active as a DNA helicase [14]. Further work is required to assign RecD protein to a specific DNA repair pathway because conflicting data have been published concerning the in vivo role of RecD in radioresistance [15][16]. D. radiodurans possesses homologs of the key components of the RecF pathway: RecJ (DR1126), RecQ (DR1289), RecF (DR1089), RecO (DR0819), and RecR (DR0198) suggesting that the RecF pathway is the main recombinational repair pathway in this organism, as observed in other bacteria that lack RecBCD homologs [13]. D. radiodurans also lacks homologs of the SbcB nuclease, an inhibitor of the RecF pathway in E. coli. Moreover, it was shown that expression in trans of the SbcB protein from E. coli renders D. radiodurans cells radiation-sensitive [17].

In this paper, we investigate the role of the D. radiodurans proteins belonging to the RecF pathway in ESDSA and/or homologous recombination. We demonstrate that RecJ exonuclease is an essential protein for cell viability. We show that the RecF, RecO, RecR proteins as well as the RecA protein are absolutely required for massive DNA synthesis during DSB repair whereas RecQ appears to be substituted by the UvrD helicase to play a role in this process. We propose that RecJ, in conjunction with UvrD, could generate the single stranded tails on which RecFOR will stimulate RecA loading. Interestingly, an intact genome could be slowly reconstituted in the absence of RecA, RecF, RecO or RecR, suggesting alternate DSB repair through non-homologous end joining (NHEJ) and/or single-strand annealing (SSA).

Results

recJ is an essential gene in D. radiodurans

To determine the importance of the RecFOR pathway in DSB repair and radioresistance in D. radiodurans, we replaced the coding regions of key genes belonging to this pathway (recJ, recQ, recF, recO, and recR) with an antibiotic resistance cassette. The deletion-substitution alleles were constructed in vitro using the tripartite ligation method [18] and introduced by transformation into D. radiodurans to replace the corresponding wild-type alleles via homologous recombination. Because D. radiodurans contains from 4 to 10 genome equivalents [19],[20], the transformants were extensively purified on selective media in order to obtain the mutant homogenotes whose purity was verified by PCR. Whereas only few rounds of purification on selective antibiotic plates sufficed to obtain ΔrecQ, ΔrecF, ΔrecO and ΔrecR homogenotes (see Figure S1), in the case of recJ, the wild-type allele was present together with the ΔrecJ allele even after seven rounds of purification of three independent candidates (Figure 1), suggesting that RecJ protein is essential for cell viability.

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Figure 1. Schematic representation and test of deletion-substitution in the D. radiodurans recJ gene.

(A) Schematic representation of the allele replacement event in recJ gene. Short arrows indicate the position of specific primers used for diagnostic PCR. Primers are described in Table S1. (B) PCR analysis of three independent candidate recJ mutants and three independent ΔrecJ (prepUTS-recJ+) mutants.

https://doi.org/10.1371/journal.pgen.1000774.g001

To obtain positive evidence for the essentiality of the recJ gene, we used the new diagnostic assay described by Nguyen et al [21]. For this purpose, the recJ gene was cloned onto a prepUTs vector thermosensitive for replication in D. radiodurans [21]. The sequence of DR1126 (recJ) in strain ATCC 13939 (GenBank, accession number QG856645) was found to differ from the DR1126 published sequence [22]. An additional G was found 7 nucleotides upstream the published putative GTG initiation codon and another additional G was found 58 nucleotides before the published putative TGA STOP codon giving rise to a RecJ protein containing 705 aa (versus 684 aa in the RecJ protein predicted from the previously published sequence) with 64 additional amino acids in the N-terminal domain and 43 aa missing in the C-terminal domain of the protein. The predicted sequence of the RecJ protein in strain ATCC 13939 displays a better alignment with the published protein sequences of the E. coli, Deinococcus geothermalis and Thermus thermophilus RecJ proteins (Figure S2). The recombinant plasmid was introduced into a recJ+ recipient and the chromosomal copy of recJ in the resulting merodiploid strain was inactivated (Figure 1). If recJ is an essential gene, the cells will die upon loss of the complementing plasmid at the non permissive temperature. As can be observed in Figure 2 (lanes 1–3), the ΔrecJ (prepUTs-recJ+) bacteria grew normally at 28° (the permissive temperature for the plasmid) but lose viability at 37° (the non-permissive temperature for the plasmid), demonstrating the essentiality of the recJ gene.

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Figure 2. recJ is essential for D. radiodurans viability.

Strains were grown in liquid medium with spectinomycin at 28°C. The dilutions of cells were spotted on medium with or without spectinomycin at 28°C (A) or 37°C (B). Lane 1–3: strain GY14110 [ΔrecJ (prepUTs-recJ+)], lane 4: strain GY13781 containing thermosensitive plasmid p13840 (prepUTs), lane 5: strain GY13786 containing non-thermosensitive plasmid p11554 (prepU).

https://doi.org/10.1371/journal.pgen.1000774.g002

Sub-lethal phenotype of ΔrecF, ΔrecO, and ΔrecR mutants

The ΔrecF, ΔrecO, and ΔrecR mutants, though viable, showed a greatly impaired growth. Indeed, the mutants had a generation time 4-fold longer than the wild-type (5 hours for the mutants versus 80 min for the wild-type) and comparable to that of a ΔrecA mutant. Furthermore, cells devoid of RecF, RecO or RecA proteins had a 10-fold reduced plating efficiency as compared to the wild-type strain and this defect was even more pronounced in the ΔrecR mutant, displaying a 30-fold reduced plating efficiency (Figure 3).

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Figure 3. D. radiodurans cells devoid of recA, recF, recO, or recR genes show reduced plating efficiency.

Bacterial strains GY12965 (ΔrecF, open circles), GY 12966 (ΔrecO, open squares), GY 12967 (ΔrecR, filled triangles), GY 12968 (ΔrecA, filled circles), and wild-type (closed squares) were incubated at 30°C. At different times, incubation samples were taken and the A650 values of the cultures and the numbers of viable cells/ml were measured.

https://doi.org/10.1371/journal.pgen.1000774.g003

UvrD helicase is required for rapid repair of DNA double-strand breaks whereas RecQ and RecD helicases are dispensable

In E. coli, the RecQ helicase initiates DSB repair via the RecFOR pathway by unwinding duplex DNA in the 3′-5′ direction, while the single stranded DNA exonuclease RecJ hydrolyzes the 5′ strand to provide a DNA-substrate for RecA loading onto the 3′ strand [4],[23].

We found that inactivation of the RecQ helicase in D. radiodurans had no effect on radioresistance, because the knockout mutant was as resistant to γ-irradiation as the wild-type strain (Figure 4A). This result suggests that other helicase(s) might be involved in the initiation step of DSB repair in this organism. We tested the RecD and UvrD helicases for putative roles in DSB repair. We found that a ΔrecD deletion mutant was as radioresistant as the wild-type strain, whereas a ΔuvrD mutant showed a reduction in survival that ranged from 5-fold at 11.6 kGy to more than 100-fold at 17.8 kGy (Figure 4A). However, the mutant still retained significant radioresistance as compared to a repair-deficient ΔrecA strain (Figure 4B), suggesting that other helicase(s) may overlap in function with UvrD and thus lessen the effect of a ΔuvrD mutation. To test this hypothesis, we investigated whether the combined absence of UvrD and RecQ or UvrD and RecD proteins results in a more dramatic effect on radio-resistance. As seen in Figure 4A, the ΔuvrD ΔrecQ double mutant bacteria were not more sensitive to γ-rays than a ΔuvrD single mutant. In contrast the ΔuvrD ΔrecD double mutant bacteria were slightly more sensitive to γ-rays than a ΔuvrD single mutant, suggesting that the RecD helicase may have a partial back-up function in the absence of UvrD.

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Figure 4. Increased sensitivity to γ-irradiation of cells devoid of RecF, RecO, RecR, or UvrD.

(A) Increased sensitivity of cells devoid of uvrD gene. R1 (wild-type, open inverted triangles), GY12957 (ΔrecQ, filled squares), GY12974 (ΔuvrD, filled circles), GY12975 (ΔrecQΔuvrD, open squares), GY12976 (ΔrecDΔuvrD, filled diamonds), GY12977 (ΔrecQΔrecD, filled triangles), GY13130 (ΔrecD, open circles) bacteria were exposed to γ-irradiation at doses indicated on the abscissa; and cell survival was measured as described in the Materials and Methods. (B) ΔrecFOR mutants are as sensitive as ΔrecA mutant to γ-irradiation. Bacterial strains GY12936 (wild-type/p11520, inverted triangles), GY14115 (ΔrecA/p11559, filled circles), GY14116 (ΔrecO/p11520, filled diamonds), GY14117 (ΔrecF/p11520, filled squares), GY14118 (ΔrecR/p11520, filled triangles), GY14111 (ΔrecA/p11562: recA+, open circles), GY14112 (ΔrecO/p11860: recO+, open diamonds), GY14113 (ΔrecF/p11862: recF+, open squares), GY14114 (ΔrecR/p11870: recR+, open triangles) were exposed to γ-irradiation at doses indicated on the abscissa, and cell survival was measured as described in the Materials and Methods.

https://doi.org/10.1371/journal.pgen.1000774.g004

To investigate the possible role(s) of the UvrD helicase in DSB repair, we examined whether the ΔuvrD mutant was affected in two key steps of the ESDSA pathway: (i) the reassembly of broken DNA fragments and (ii) the associated massive DNA synthesis. Cells were exposed to 6.8 kGy γ-irradiation, a dose that introduces approximately 200 DSB per genome equivalent in a D. radiodurans cell [24]. Recovery from DNA damage was monitored by the appearance of the complete pattern of the 11 resolvable genomic DNA fragments generated by NotI digestion [25] and de novo DNA synthesis was measured by labelling DNA with a 15 min 3H-TdR pulse at different times post irradiation. As seen in Figure 4, ΔrecQ and ΔrecD cells repaired DSB with the same kinetics as the wild-type strain, reconstituting an intact genome within 3 h post-irradiation (Figure 5A). In contrast, in ΔuvrD bacteria, this process required approximately 8 h (Figure 5A), the kinetics of DSB repair had an increased latent phase (240 min in the mutant versus 90 min in the wild-type) during which DNA degradation took place and a slower rate of fragment assembly. Moreover, resumption of DNA synthesis was delayed in ΔuvrD mutant bacteria and its rate was 2-fold lower than that observed in wild-type bacteria (Figure 5B). These results suggest that UvrD plays a major role in DSB repair through ESDSA.

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Figure 5. DNA repair and DNA synthesis in ΔrecQ, ΔrecD, and ΔuvrD mutants.

(A) Kinetics of DSB repair in wild-type, ΔrecQ, ΔrecD, and ΔuvrD mutants followed by pulsed-field gel electrophoresis (PFGE). PFGE shows NotI treated DNA from unirradiated cells (lane pre-irradiation) and from irradiated cells (6,8 kGy) immediately after irradiation (0) and at the indicated incubation times (hours). (B) Rate of DNA synthesis in wild-type, ΔrecQ, ΔrecD, and ΔuvrD mutants. Incorporation of [3H]thymidine during 15-min pulse labelling measures the global rate of DNA synthesis in 6.8 kGy-irradiated (filled circles) and unirradiated (open circles) bacteria.

https://doi.org/10.1371/journal.pgen.1000774.g005

A major role of RecFOR in D. radiodurans radioresistance

The ΔrecF, ΔrecO and ΔrecR mutants were as radiosensitive as a ΔrecA mutant (Figure 4B). The radiosensitivity of the ΔrecF and ΔrecO mutants was fully complemented by a plasmid expressing RecF or RecO proteins in trans, whereas, in the case of the ΔrecR mutant, bacteria expressing recR+ in trans only recovered 90% of wild-type survival after γ-irradiation (Figure 4B). Because recR belongs to a putative operon, the radiosensitivity of the knock-out mutant may be due in part to a polar effect of our construct on a downstream gene or to a sub- or overoptimal plasmid-based expression of the RecR protein.

The kinetics of DNA double-strand-break repair in the three mutants was very similar to that observed in a ΔrecA mutant (Figure 6A). There was a slight and progressive reassembly of the radiation-induced DNA fragments that culminates at 24h post-irradiation incubation in the restitution of a complete pattern of the 11 NotI resolvable fragments (Figure 6A). However, only very faint bands of reconstituted chromosome were observed 24h post-irradiation incubation suggesting that a complete genome was only present in a small subpopulation of the mutant cells. The initial degradation of the damaged DNA that can be seen in the wild-type during the first hour of post-irradiation incubation (Figure 5A) was also markedly reduced in the three recFOR mutants (Figure 6A), as was previously observed for a ΔrecA mutant [2]; Figure 6A).

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Figure 6. DNA repair and DNA synthesis in ΔrecA, ΔrecF, ΔrecO, and ΔrecR mutants.

(A) Kinetics of DSB repair in wild-type, ΔrecA, ΔrecF, ΔrecO, and ΔrecR mutants. PFGE shows NotI treated DNA from unirradiated cells (lane pre-irradiation) and from irradiated cells (6,8kGy) immediately after irradiation (0) and at the indicated incubation times (hours). (B) Rate of DNA synthesis in wild-type, ΔrecA, ΔrecF, ΔrecO, and ΔrecR mutants. Incorporation of [3H]thymidine during 15-min pulse labelling measures the global rate of DNA synthesis in irradiated (filled circles) and unirradiated (circles) bacteria.

https://doi.org/10.1371/journal.pgen.1000774.g006

The reconstitution of the complete genomic NotI pattern in the irradiated recFOR mutants did not result from the multiplication of rare survivors, because there was no observable increase in the number of CFU during 24 hours of incubation of irradiated cells (data not shown). Pulses of 3H-TdR showed that no DNA synthesis was observed during the 6 hours following γ-irradiation (Figure 6B) nor during the late fragment assembly in ΔrecF, ΔrecO and ΔrecR bacteria (data not shown), as observed previously in ΔrecA bacteria [2]. Moreover, the late genome reconstitution in these mutants is not sufficient to ensure cell survival.

In conclusion, our results suggest that RecF, RecO and RecR proteins, like RecA protein, play a central role in Deinococcal radioresistance, probably because they are absolutely required for loading RecA onto its DNA substrate to perform efficient double-strand-break repair via ESDSA and recombinational repair pathways.

Discussion

Recent studies have shown that the ability of D. radiodurans to cope with the DNA-shattering effect of elevated doses of γ-irradiation or dessication involves a robust DNA repair process called extended synthesis-dependent strand annealing (ESDSA) in which long tracts of newly synthesized DNA are made [1],[2]. Whereas the dependence of massive DNA synthesis on Pol I, Pol III and RecA (or its homolog RadA) in ESDSA is well documented [2], little is known about the cellular factors required for the initial steps of this process: (i) the formation of the single stranded 3′ overhangs and (ii) the loading of RecA on this recombinogenic substrate to prime DNA synthesis.

Major role of RecFOR proteins in DNA double-strand-break repair in Deinococcus radiodurans

D. radiodurans is naturally devoid of RecB and RecC proteins but contains homologs of key proteins of the E. coli RecF pathway: RecJ, RecQ, RecF, RecO and RecR. We found that cells devoid of RecF, RecO or RecR proteins were as radiosensitive as cells devoid of RecA. The ΔrecF, ΔrecO and ΔrecR mutants, as previously shown for a ΔrecA mutant [2], supported a slow and progressive reassembly of the radiation-induced DNA fragments. As in ΔrecA cells, genome reassembly was not accompanied by significant DNA synthesis, suggesting that cells devoid of RecF, RecO or RecR proteins are deficient for ESDSA, with repair of DSB probably mediated by RecA-independent pathways, such as single-strand annealing (SSA) or non-homologous end joining (NHEJ). The mutants also showed an important lethal sectoring during normal growth, similar to that observed in a ΔrecA mutant, in which about 90% of the visible cells failed to give rise to colonies [26]. The similarity of the ΔrecFOR and ΔrecA phenotypes supports the hypothesis that RecA activity in D. radiodurans is totally dependent on a functional RecF pathway.

Following exposure of D. radiodurans to ionizing radiation, there is a rapid and extensive degradation of chromosomal DNA that plays an important role in the repair process in this species (reviewed by [24]). The initial degradation of damaged DNA can be observed using pulsed-field electrophoresis as a reduction of the amount of the double stranded DNA fragments during the first 90 min of post-irradiation incubation in the wild-type cells, prior to the onset of fragment assembly. Slade et al observed that DNA degradation is markedly reduced in a ΔrecA mutant, leading the authors to propose that RecA itself regulates maturation of double-strand ends by controlling both DNA degradation and DNA synthesis [2]. We found that DNA degradation was also reduced in ΔrecF, ΔrecO or ΔrecR mutants, as well as in a ΔrecA mutant. RecA may play a regulatory role in the control of expression of nuclease-like activities in response to DNA damage, while RecFOR proteins may be indirectly involved in DNA degradation by facilitating the formation of the RecA filament on single stranded DNA. It would be interesting to analyse DNA degradation in the Deinococcal recA424 mutant, which retains the RecA coprotease activity while remaining deficient in recombination activity [27].

Biochemical studies using RecFOR proteins from E. coli indicate that these proteins act together as mediators of the formation of the pre-synaptic RecA filament onto single stranded DNA. Current models agree on the formation of two complexes, RecFR and RecOR. RecOR is generally thought to be responsible for rendering SSB-coated ssDNA accessible to RecA. RecFR targets dsDNA or dsDNA-ssDNA junctions and is responsible for the targeting of RecA to the ssDNA region of gaps [28][31]. More recently, it was proposed that RecR is the key component with which RecA interacts, whereas the RecO protein can displace SSB and bind to single stranded DNA independently of RecR, yet does not load RecA until RecR is added [32],[33]. When RecF is present, a RecFOR loading pathway, independent of RecO-SSB interactions, is preferred [33].

Recent X-ray structural analysis of RecO and RecR proteins from D. radiodurans confirms the existence of a RecOR complex in this organism. RecR molecules form a ring structure that can encircle both dsDNA and ssDNA [34],[35]. The structure of the RecF protein from D. radiodurans has also recently been elucidated, showing that the RecF protein exhibits extensive structural similarity with the head domain of the eukaryotic Rad50 protein [36]. More recently, a model of recognition of the ds-DNA ss-DNA junction in D. radiodurans through a DNA-protein and protein-protein interaction was proposed: RecR interacts with ssDNA coated by RecO-SSB, which leads to the elevation of the local concentration of RecR and stimulates RecF binding in the adjacent ds-DNA [37].

Essentiality of RecJ protein

While inactivation of RecA or RecFOR proteins in D. radiodurans reduced cell viability, inactivation of RecJ resulted in a fully lethal phenotype. In other bacterial species, mutations in recJ are highly synergistic with those in recBCD. In E. coli, recBC recJ mutants are recombination deficient, extremely UV-sensitive and highly growth disrupted [11],[38]. In Salmonella typhimurium, recB recJ mutants also display a similar phenotype [39]. More recently, it was shown that a recJ knock-out is colethal with recBCD or recD deletions in Acinetobacter baylyi [40]. The strongly reduced viability (or lethality) of recBC recJ bacteria was attributed to severe deficiencies in repair of spontaneous DNA damage and inactivated replication forks [39],[40]. It should be noted that, whereas E. coli and S. typhimurium contain at least three 5′-3′ exonucleases [RecJ, Exo V (RecBCD), Exo VII (XseAB)], the genome of A. baylyi encodes only Exo V and RecJ, and that of D. radiodurans encodes only RecJ and one of the two subunits of Exo VII. We propose that Exo VII has some back-up activity in E. coli or S. typhimurium when RecJ and ExoV are inactivated, an activity that is missing in A. baylyi and D. radiodurans.

In E. coli, RecJ and RecFOR were proposed to be required to restore DNA synthesis after UV-induced damage [41],[42]. The mechanism by which lesion-blocked replication forks recover in E. coli is thought to involve the formation of reverse replication fork intermediate stabilized by RecA and RecF and degraded by the RecQ-RecJ helicase-nuclease when RecA or RecF are absent [41]. The fork regression allows DNA repair enzymes to remove the blocking lesion, thus restoring processive replication. In the absence of RecJ, the recovery of replication is significantly delayed and both replication recovery and cell survival become dependent on translesion synthesis by DNA polymerase V [42]. D. radiodurans does not encode a bypass DNA polymerase belonging to the Y family, and under these conditions RecJ may be essential for restoration of replication forks after arrest, even in cells not treated by DNA damaging agents. Frequent DNA double-strand-breaks were thought to arise spontaneously ranging from 0.2–1 per genome replication in E. coli [4],[43]. However, a more direct quantification of DNA double-strand-breaks indicated that the rate of spontaneous breakage is 20 to 100-fold lower than predicted, only one percent of the cells having one or more DNA double-strand-breaks per genome replication [44].

Because cells devoid of RecA or RecFOR are viable, the ΔrecJ lethal phenotype cannot be only due to a possible deficiency in DSB repair, leading us to postulate that RecJ is required in D. radiodurans for more than one cellular process and that inactivation of all of these processes (DSB repair, fork reversion, restoration of a fork structure after regression …) may be lethal for the cell.

Involvement of UvrD in DNA double-strand-break repair

In E. coli, the RecJ exonuclease has been mainly associated with the RecQ helicase in recombination and repair (see, for review, [4]). The RecQ protein from D. radiodurans shows unusual domain architecture with three tandem HRDC (Helicase RNase D C-terminal) domains in addition to the conserved helicase and RQC (RecQ C-terminal) domains. The tandem arrangement of HRDC domains regulates the specificity of the binding of RecQ to DNA substrates [45],[46]. Here, we found that ΔrecQ mutants displayed a wild-type level of resistance to γ-irradiation, exhibiting the same kinetics as the wild-type strain for fragment reassembly and DNA synthesis after irradiation. In another report, a recQ knock-out mutant was shown to be highly sensitive to H2O2 and slightly more sensitive than the wild-type strain to elevated γ-irradiation doses [46]. It was recently proposed that recQ deletion, by causing transcriptome alteration, would generate ROS accumulation and Fe and Mn alterations [47]. Our findings suggest that the RecQ helicase in D. radiodurans plays only a minor role in DSB repair, probably as consequence of redundant functions provided by other helicase(s). Mutants devoid of RecD behave like ΔrecQ mutants in that they show wild-type radioresistance and repair capacity. The Deinococcal RecD protein has been characterized in vitro as a helicase with 5′-3′ polarity (opposite to that of RecQ) and low processivity [14].

In contrast, we found that inactivation of UvrD (helicase II) markedly reduced Deinococcal radioresistance and severely delayed the kinetics of DSB repair. UvrD has been largely characterized for its role in nucleotide excision repair (NER) and mismatch repair (MMR) in E. coli (reviewed by [48]). However, the altered kinetics of repair and the radiosensitivity of ΔuvrD bacteria are unlikely to result from a deficiency in the NER pathway because uvrA deficient mutant bacteria display a wild-type survival pattern following exposure to ionizing-radiation (Figure S3). The ΔmutS bacteria deficient for MMR were also shown to be as radioresistant as wild-type bacteria [18]. Interestingly, the delayed kinetics of DSB repair in cells devoid of UvrD coincided with DNA synthesis (albeit significantly less extensive than that observed in the wild-type cells) suggesting that ESDSA repair could take place but only inefficiently in this mutant. We propose that UvrD is involved in ESDSA and that the redundant activity of other helicase(s) is responsible for the residual DNA repair capacity observed in the ΔuvrD mutant. The fact that the ΔrecQΔuvrD and the ΔrecDΔuvrD double mutant bacteria were not as radiosensitive as ΔrecA bacteria suggests that neither RecQ nor RecD can solely fulfil this role, and that other helicase(s) may be involved. Helicase IV (HelD) has been implicated as partner of the RecJ exonuclease in the RecF pathway in E. coli, together with Helicase II and RecQ [49]. Mutational inactivation of Helicase IV has no effect on the radioresistance of D. radiodurans [50]. Alternatively, RecA itself, by binding to double stranded DNA ends, could unwind DNA and provide a DNA substrate for RecJ or another 5′-3′ exonuclease. Indeed, in vitro, the D. radiodurans RecA protein binds preferentially to double stranded DNA [51].

In E. coli, the UvrD protein was not shown to be required for DNA double-strand-break repair. In contrast, it was shown to possess an anti-recombination activity, which has been related to its capacity to disrupt the RecA nucleoprotein filament [52],[53]. This activity is conserved among many species [54]. Thus, as in other species, the D. radiodurans UvrD protein might not be involved directly in the maturation of DNA double-strand ends. Several observations suggest that E. coli UvrD may be involved in DNA replication [55][58] and it was shown to be required for DNA replication of several different rolling-circle plasmids in E. coli [59]. Thus, the D. radiodurans UvrD protein might also act in the DNA synthesis step of ESDSA.

A scenario for DNA double-strand-break repair through ESDSA in D. radiodurans

Taking into account our results and those of others [2],[60],[61], we propose a model for the role of the proteins of the RecF pathway in ESDSA (Figure 7). In this model, RecJ or an as-yet unidentified exonuclease associated with the UvrD helicase, could generate 3′ single stranded DNA ends required for priming of massive DNA synthesis. Alternatively, RecA itself, by binding to double stranded DNA ends, could unwind DNA and provide a DNA substrate for RecJ or another exonuclease.

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Figure 7. Model of initiation of DNA double-strand-break repair through ESDSA in D. radiodurans.

https://doi.org/10.1371/journal.pgen.1000774.g007

Analysis of the transcriptome of D. radiodurans revealed a large group of genes that are up-regulated in response to either desiccation or ionizing radiation [62]. The deinococcal specific ddrA (DR0423) and ddrB (DR0070) genes were found among the most highly induced in response to each stress and their inactivation promotes sensitization of the mutant cells to ionizing radiation [62]. The DdrA protein is involved in protection of 3′ DNA single stranded ends [60] and presumably ensures long-lived recombinational substrates [63]. The DdrB protein binds single stranded DNA but not duplex DNA and is the prototype of a new bacterial SSB family [61]. The induction of an alternative SSB following irradiation has potentially broad significance for efficient genome reconstitution. We propose that during initiation of ESDSA, DdrA protects the 3′ DNA ends whereas SSB or the SSB-like DdrB binds to single stranded DNA.

Our results supporting the idea that RecA activity in D. radiodurans is totally dependant on a functional RecF pathway, lead us to propose that RecFOR renders SSB or DdrB- coated single stranded DNA accessible to RecA and favors formation of a RecA nucleoprotein filament required for invasion of a double stranded homologous DNA. Finally, as described previously [2], Pol III and Pol I can promote DNA synthesis, eventually with the help of the UvrD helicase.

Moreover, the compact D. radiodurans nucleoid structure that remains unaltered after high-dose γ-irradiation may passively contribute to radioresistance by preventing the dispersion of free DNA ends [64],[65]. Such a condensed genome may provide suitable scaffolds for DNA repair through ESDSA, recombinational and/or DNA end joining processes.

In conclusion, we demonstrate the essential role of key components of the D. radiodurans RecF pathway in ESDSA. We show for the first time that (i) inactivation of only one exonuclease, RecJ, results in cell lethality (ii) cells devoid of RecF, RecO or RecR display greatly impaired growth (iii) RecF, RecO or RecR proteins are essential for radioresistance through ESDSA (iv) UvrD helicase has an unexpected crucial function in DNA double-strand-break repair through ESDSA.

Materials and Methods

Bacterial strains, cultures, media, and transformation

Bacterial strains and plasmids are listed in Table 1 and Table 2, respectively. The Escherichia coli strain DH5α was used as the general cloning host and strain SCS110 was used to propagate plasmids prior to introduction into D. radiodurans via transformation [66]. All D. radiodurans strains were derivatives of strain R1 (ATCC 13939). D. radiodurans was grown in TGY2X (1% tryptone, 0.2% dextrose, 0.6% yeast extract) or in TGYA (0.5% tryptone, 0.2% dextrose, 0.15% yeast extract) at 30°C with aeration or on TGY1X plates solidified with 1.5% agar. E. coli strains were grown in Luria-Bertani (LB) broth (Gibco Laboratories). When necessary, media were supplemented with the appropriate antibiotics used at the following final concentrations: chloramphenicol 3 µg/mL for D. radiodurans; kanamycin 6 µg/mL for D. radiodurans; tetracycline 2.5 µg/mL for D. radiodurans; hygromycin 50 µg/mL; spectinomycin 40 µg/mL for E. coli and 75 µg/mL for D. radiodurans. Transformation of D. radiodurans with PCR products, genomic DNA, or plasmids was performed as previously described [26].

DNA manipulations

Plasmid DNA was extracted from E. coli using the QIAprep spin miniprep kit (Qiagen). Chromosomal DNA of D. radiodurans was isolated as previously described [67]. Amplification of plasmid or genomic DNA by PCR was performed with DyNAzyme EXT DNA polymerase (Finnzyme) or Extensor Hi-Fidelity PCR enzyme Mix (ABgene). Oligonucleotides used are listed in Table S1.

Deletion of genes in D. radiodurans

The recF, recO, recR, recA, uvrD, recD, recQ, recJ disruption mutants were constructed by the tripartite ligation method [18]. The mutated alleles constructed in vitro were then used to transform D. radiodurans to replace their wild-type counterpart by homologous recombination. The genetic structure and the purity of the mutants were checked by PCR using primers described in Table S1.

Construction of plasmids

Plasmid p11869 is a derivative of the thermosensitive plasmid p13840 [21]. To construct p11869, the recJ gene was amplified by PCR using the primer pair (PS441/PS442) and the product was cloned into plasmid p13840 between the NdeI/XhoI sites.

Plasmids p11862, p11860 and p11870 carrying the recF, recO, recR genes, respectively, under the control of their natural promoter were used to express the recF, recO, recR genes in a ΔrecF, ΔrecO, ΔrecR background. To construct plasmid p11860, the recO gene was amplified by PCR using the primer pair (PS402/PS403) and the resultant product was cloned into plasmid p11520 [68] between the SacI/BamHI sites. Plasmid p11870, containing the recR gene, was constructed in a similar manner using the primer pairs PS414/PS415. The recF gene was cloned into plasmid p11520 between the SpeI/BglII sites in a similar manner using the primers PS410/PS411 to obtain p11862. All constructions were verified by DNA sequencing.

Plasmid p11562 [63], expressing recA from a PSpac promoter, was used to transform GY12968: ΔrecAΩkan giving rise to strain GY14111. The expression of recA was induced by adding 10 mM IPTG to the media.

Treatment of D. radiodurans with γ-irradiation

Exponential cultures, grown in TGY2X (supplemented with spectinomycin when necessary), were concentrated to an A650 = 10 in TGY2X and irradiated on ice with a 137Cs irradiation system (Institut Curie, Orsay, France) at a dose rate of 44.7 Gy/min. Following irradiation, diluted samples were plated on TGY plates. Colonies were counted after 3–4 days incubation at 30°C.

Assay of recJ gene essentiality

The essentiality of genes was evaluated in a growth experiment in which the strains grown at 28°C in liquid medium with spectinomycin, were serially diluted, plated on TGY agar and incubated at 28°C or 37°C in the presence or absence of spectinomycin [21].

Kinetics of DNA repair measured by pulse-field gel electrophoresis

Non-irradiated or irradiated (6.8 kGy) cultures were diluted in TGY2X to an A650 = 0.2 and incubated at 30°C. At different post-irradiation recovery times, culture aliquots (5mL) were removed to prepare DNA plugs as described previously [60]. The agarose embedded DNA plugs were digested for 16 h at 37°C with 10 units of NotI restriction enzyme. After digestion, the plugs were subjected to pulsed field gel electrophoresis as described previously [69].

Rate of DNA synthesis measured by DNA pulse labelling

The rate of DNA synthesis was measured according to a modified protocol from Zahradka et al [1]. Exponential cultures, grown in TGYA, were concentrated to an A650 = 20 in TGYA and irradiated as described previously. Non-irradiated or irradiated cultures (6.8 kGy) were diluted in TGYA to an A650 = 0.2 and incubated at 30°C. At different time 0.5mL samples were taken and mixed with 0.1mL pre-warmed TGYA containing 4.8 µCi [methyl-3H]thymidine (PerkinElmer, specific activity 70–90 Ci/mmol). Radioactive pulses of 15 min were terminated by addition of 2 mL ice-cold 10% TCA. Samples were kept on ice for at least 1 h, and then collected by vacuum filtration onto Whatman GF/C filters followed by washing twice with 5mL 5% TCA and twice with 5mL 96% ethanol. Filters were dried for 10 min under a heat source and placed in 4 mL scintillation liquid. The precipitated counts were measured in a liquid scintillation counter (Packard, TRI- carb 1600 TR).

Supporting Information

Figure S1.

Schematic representation and test of deletion-substitution of D. radiodurans recF, recO, recR, and recQ genes. (A) schematic representation of the allele replacement event of recF (A.1), recO (A.2), recR (A.3), and recQ (A.4) genes. Short arrows indicate the position of specific primers used for diagnostic PCR. Primers are described in Table S1. (B) PCR analysis of ΔrecF (B.1), ΔrecO (B.2), ΔrecR (B.3), and ΔrecQ (B.4) mutants.

https://doi.org/10.1371/journal.pgen.1000774.s001

(0.72 MB TIF)

Figure S2.

Alignment of the D. radiodurans ATCC13939 RecJ protein. Alignment of the D. radiodurans ATCC13939 RecJ protein with its TIGER sequence and the corresponding Deinococcus geothermalis, Termus thermophilus, and E. coli RecJ protein. The alignment was generated using Clone Manager program. Shading was based on amino acid identity (green boxes).

https://doi.org/10.1371/journal.pgen.1000774.s002

(0.04 MB DOC)

Figure S3.

Nucleotide Excision Repair deficient uvrA bacteria are as radioresistant as the wild type. R1 (wild type, open inverted triangles), GY12974 (ΔuvrD, filled circles), and GY9614 (uvrA1, filled squares) bacteria were exposed to γ-irradiation at doses indicated on the abscissa, and cell survival was measured as described in the Materials and Methods.

https://doi.org/10.1371/journal.pgen.1000774.s003

(0.10 MB TIF)

Table S1.

Overview of primers used for construction of mutant strains, cloning, and diagnostic PCR experiments.

https://doi.org/10.1371/journal.pgen.1000774.s004

(0.04 MB DOC)

Acknowledgments

Adriana Bailone is acknowledged for critical reading of the manuscript and Bénédicte Michel for very helpful discussions. We thank John Battista for discussions on the role of helicases in radioresistance, Michael DuBow for help with English, Issay Narumi for the gift of the pKatHPH4 plasmid, the Institut Curie for the use of the 137Cs irradiation system, and Vincent Favaudon for help with γ-irradiation.

Author Contributions

Conceived and designed the experiments: EB PS SS. Performed the experiments: EB PS GC. Analyzed the data: EB PS SS. Wrote the paper: EB PS SS.

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