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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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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.
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pgen-1000774-g006: 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.

Mentions: 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).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000774-g006: 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.
Mentions: 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).

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