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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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Inactivation of xonA and recQ genes does not affect UV radiation survival (A) and γ-survival (B) of nuclease deficient but RecA loading proficient recB1080 recD mutants.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080 recD; (▴) recB1080 recD ΔxonA; (▾) recB1080 recD ΔrecQ; (♦) recB1080 recD ΔrecQ ΔxonA.
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pone-0039030-g003: Inactivation of xonA and recQ genes does not affect UV radiation survival (A) and γ-survival (B) of nuclease deficient but RecA loading proficient recB1080 recD mutants.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080 recD; (▴) recB1080 recD ΔxonA; (▾) recB1080 recD ΔrecQ; (♦) recB1080 recD ΔrecQ ΔxonA.

Mentions: When the RecB1080CD and RecB1067CD mutant enzymes, devoid of both nuclease and RecA loading activities [14], [30], lack the RecD subunit (in recD derivatives of recB1080 and recB1067), they gain the ability to constitutively load RecA protein, while remaining nuclease deficient [14], [30]. We used the recD recB1080 and recD recB1067 mutants to determine which of the two missing RecBCD functions is/are bypassed with the functions of ExoI and RecQ helicase. When either single ΔxonA or ΔrecQ mutations, or both of them, were introduced into a recD derivative of the recB1080 mutant, they did not affect its UV and γ-survival (Figure 3). The same behavior was observed in the recD recB1067 mutant; introduction of either the ΔxonA or the ΔrecQ or both of these mutations did not influence its UV and γ-survival (data not shown). These results corroborate earlier findings that recQ[31] or xonA[32] mutation does not affect UV and γ-survival in recD derivatives of recB nuclease deficient strains. An individual ΔxonA or ΔrecQ mutation, as well as their combination, also did not change UV and γ-survival of RIK144, a recD derivative of the wt strain (not shown). These results show that ExoI and RecQ helicase are not required for DNA repair in the nuclease deficient but RecA loading proficient recD recB mutants; meaning that RecB1080C-catalyzed RecA loading at DNA ends renders the two enzymes dispensable.


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

Ivanković S, Đermić D - PLoS ONE (2012)

Inactivation of xonA and recQ genes does not affect UV radiation survival (A) and γ-survival (B) of nuclease deficient but RecA loading proficient recB1080 recD mutants.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080 recD; (▴) recB1080 recD ΔxonA; (▾) recB1080 recD ΔrecQ; (♦) recB1080 recD ΔrecQ ΔxonA.
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Related In: Results  -  Collection

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

pone-0039030-g003: Inactivation of xonA and recQ genes does not affect UV radiation survival (A) and γ-survival (B) of nuclease deficient but RecA loading proficient recB1080 recD mutants.Fraction survival is given as a fraction of the unirradiated control. Symbols: (▪) recB1080 recD; (▴) recB1080 recD ΔxonA; (▾) recB1080 recD ΔrecQ; (♦) recB1080 recD ΔrecQ ΔxonA.
Mentions: When the RecB1080CD and RecB1067CD mutant enzymes, devoid of both nuclease and RecA loading activities [14], [30], lack the RecD subunit (in recD derivatives of recB1080 and recB1067), they gain the ability to constitutively load RecA protein, while remaining nuclease deficient [14], [30]. We used the recD recB1080 and recD recB1067 mutants to determine which of the two missing RecBCD functions is/are bypassed with the functions of ExoI and RecQ helicase. When either single ΔxonA or ΔrecQ mutations, or both of them, were introduced into a recD derivative of the recB1080 mutant, they did not affect its UV and γ-survival (Figure 3). The same behavior was observed in the recD recB1067 mutant; introduction of either the ΔxonA or the ΔrecQ or both of these mutations did not influence its UV and γ-survival (data not shown). These results corroborate earlier findings that recQ[31] or xonA[32] mutation does not affect UV and γ-survival in recD derivatives of recB nuclease deficient strains. An individual ΔxonA or ΔrecQ mutation, as well as their combination, also did not change UV and γ-survival of RIK144, a recD derivative of the wt strain (not shown). These results show that ExoI and RecQ helicase are not required for DNA repair in the nuclease deficient but RecA loading proficient recD recB mutants; meaning that RecB1080C-catalyzed RecA loading at DNA ends renders the two enzymes dispensable.

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