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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 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.
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pgen-1000774-g005: 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.

Mentions: 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.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000774-g005: 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.
Mentions: 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.

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