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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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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.
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pgen-1000774-g004: 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.

Mentions: 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.


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)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806897&req=5

pgen-1000774-g004: 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.
Mentions: 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.

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