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A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.

Bentchikou E, Servant P, Coste G, Sommer S - PLoS Genet. (2010)

Bottom Line: Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation.Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD 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.

View Article: PubMed Central - PubMed

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 DeltarecA mutant: DeltarecFOR 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 gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD 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 DeltauvrD 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.

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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).
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pgen-1000774-g002: 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).

Mentions: 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.


A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans.

Bentchikou E, Servant P, Coste G, Sommer S - PLoS Genet. (2010)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806897&req=5

pgen-1000774-g002: 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).
Mentions: 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.

Bottom Line: Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a DeltarecA mutant: DeltarecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation.Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of DeltauvrD 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.

View Article: PubMed Central - PubMed

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 DeltarecA mutant: DeltarecFOR 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 gamma-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, DeltauvrD 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 DeltauvrD 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.

Show MeSH
Related in: MedlinePlus