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DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli.

Ivanković S, Đermić D - PLoS ONE (2012)

Bottom Line: Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events.The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination.Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.

ABSTRACT
Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.

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ΔxonA and ΔrecQ mutations impair DNA repair in UV irradiated (A) and γ-irradiated (B) recB1080 mutant cells, which lack nuclease and RecA loading activities of RecBCD.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080; (▴) ΔxonA recB1080; (▾) ΔrecQ recB1080; (♦) ΔxonA ΔrecQ recB1080; (•) recB268::Tn10.
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pone-0039030-g001: ΔxonA and ΔrecQ mutations impair DNA repair in UV irradiated (A) and γ-irradiated (B) recB1080 mutant cells, which lack nuclease and RecA loading activities of RecBCD.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080; (▴) ΔxonA recB1080; (▾) ΔrecQ recB1080; (♦) ΔxonA ΔrecQ recB1080; (•) recB268::Tn10.

Mentions: By using P1 transduction we introduced a recQ deletion into recB1080 and recB1067 mutants, as well as in wt strain, and assessed their DNA repair capacity. DNA repair was evaluated by measuring UV and γ-survival of the mutants. As shown in Figure 1, the recB1080 ΔrecQ mutant was considerably more sensitive to both UV and γ-irradiation than the parental recB1080 strain. At the highest UV and γ-dose tested, the survival of the double mutant was about 10 fold lower than that of the parent strain. A Δ recQ derivative of the recB1080 mutant was about two fold more radiosensitive than recB1080, when comparing survival slopes (Figure 1B). The same effect was observed in recB1067 background (not shown). Although our results are somewhat similar to those of a previous study, the conclusion of that study (that RecQ is “not needed for RecB1080CD-mediated recombination”) [16] is opposite to our conclusions. As expected, and in accord with previous studies [27], [28], RecQ inactivation did not produce any effect on UV and γ-survival of otherwise wt cells (Figure 2).


DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli.

Ivanković S, Đermić D - PLoS ONE (2012)

ΔxonA and ΔrecQ mutations impair DNA repair in UV irradiated (A) and γ-irradiated (B) recB1080 mutant cells, which lack nuclease and RecA loading activities of RecBCD.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080; (▴) ΔxonA recB1080; (▾) ΔrecQ recB1080; (♦) ΔxonA ΔrecQ recB1080; (•) recB268::Tn10.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375238&req=5

pone-0039030-g001: ΔxonA and ΔrecQ mutations impair DNA repair in UV irradiated (A) and γ-irradiated (B) recB1080 mutant cells, which lack nuclease and RecA loading activities of RecBCD.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080; (▴) ΔxonA recB1080; (▾) ΔrecQ recB1080; (♦) ΔxonA ΔrecQ recB1080; (•) recB268::Tn10.
Mentions: By using P1 transduction we introduced a recQ deletion into recB1080 and recB1067 mutants, as well as in wt strain, and assessed their DNA repair capacity. DNA repair was evaluated by measuring UV and γ-survival of the mutants. As shown in Figure 1, the recB1080 ΔrecQ mutant was considerably more sensitive to both UV and γ-irradiation than the parental recB1080 strain. At the highest UV and γ-dose tested, the survival of the double mutant was about 10 fold lower than that of the parent strain. A Δ recQ derivative of the recB1080 mutant was about two fold more radiosensitive than recB1080, when comparing survival slopes (Figure 1B). The same effect was observed in recB1067 background (not shown). Although our results are somewhat similar to those of a previous study, the conclusion of that study (that RecQ is “not needed for RecB1080CD-mediated recombination”) [16] is opposite to our conclusions. As expected, and in accord with previous studies [27], [28], RecQ inactivation did not produce any effect on UV and γ-survival of otherwise wt cells (Figure 2).

Bottom Line: Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events.The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination.Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Ruđer Bošković Institute, Zagreb, Croatia.

ABSTRACT
Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human oncogenesis.

Show MeSH
Related in: MedlinePlus