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Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea.

Rzechorzek NJ, Blackwood JK, Bray SM, Maman JD, Pellegrini L, Robinson NP - Nat Commun (2014)

Bottom Line: HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion.This system differs substantially from the bacterial end-resection paradigms.Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

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SAXS analysis of the HerA-NurA complex(A) and (B) Lateral and axial views respectively of the molecular envelope generated by ab initio SAXS modelling, with crystallographic models of HerA (PDB code: 4D2I) and NurA (PDB code: 2YGK) manually docked in it. The axial view is clipped at the level of the ATPase domains of the HerA hexamer, to visualise the pore-like cavity present in the SAXS envelope, which defines the position of HerA within the map. (C) Experimental SAXS curve for the HerA-NurA complex, in the momentum transfer range 0.006<q<0.156 used for analysis (q = 4π sinθ/λ, where 2θ is the scattering angle and λ is the wavelength). (D) Guinier plot for the low-q region of the SAXS data. Linear agreement of the Guinier equation (red line) with the experimental data (blue crosses) is confirmed by low residuals (red dots) within the boundaries indicated by the arrow, enabling estimation of the radius of gyration, Rg. (E) Inter-atomic pairwise distribution function, P(r), calculated by GNOM. Data were automatically truncated to qmax≈0.16 according to the 8/Rg limit rule for ab initio modelling by DAMMIF. The P(r) function smoothly approaches zero at Dmax=158.1 Å. (F) Theoretical scattering curve (red line) for 1 of 20 ab initio models generated by DAMMIF, fitted to the experimental data (green crosses). All 20 models were superimposed, averaged and filtered by DAMAVER to generate the final model used for envelope generation in (A) and (B). The mean normalized spatial discrepancy (NSD) for the 20 aligned models was 0.619±0.048, and one model was omitted from averaging as an outlier.
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Figure 8: SAXS analysis of the HerA-NurA complex(A) and (B) Lateral and axial views respectively of the molecular envelope generated by ab initio SAXS modelling, with crystallographic models of HerA (PDB code: 4D2I) and NurA (PDB code: 2YGK) manually docked in it. The axial view is clipped at the level of the ATPase domains of the HerA hexamer, to visualise the pore-like cavity present in the SAXS envelope, which defines the position of HerA within the map. (C) Experimental SAXS curve for the HerA-NurA complex, in the momentum transfer range 0.006<q<0.156 used for analysis (q = 4π sinθ/λ, where 2θ is the scattering angle and λ is the wavelength). (D) Guinier plot for the low-q region of the SAXS data. Linear agreement of the Guinier equation (red line) with the experimental data (blue crosses) is confirmed by low residuals (red dots) within the boundaries indicated by the arrow, enabling estimation of the radius of gyration, Rg. (E) Inter-atomic pairwise distribution function, P(r), calculated by GNOM. Data were automatically truncated to qmax≈0.16 according to the 8/Rg limit rule for ab initio modelling by DAMMIF. The P(r) function smoothly approaches zero at Dmax=158.1 Å. (F) Theoretical scattering curve (red line) for 1 of 20 ab initio models generated by DAMMIF, fitted to the experimental data (green crosses). All 20 models were superimposed, averaged and filtered by DAMAVER to generate the final model used for envelope generation in (A) and (B). The mean normalized spatial discrepancy (NSD) for the 20 aligned models was 0.619±0.048, and one model was omitted from averaging as an outlier.

Mentions: The structure-based mutagenesis data presented here are in agreement with the coaxial arrangement for the HerA-NurA complex predicted in our previously published study (16), where the NurA dimer docks on top of the HAS-barrel face of the HerA hexamer. We note that the exposed hydrophobic patches on the surface of NurA (16) and HerA, identified by mutagenesis, appear suitably aligned to mediate this interaction (Supplementary Fig. 7). To provide further insight into the mechanism of DNA-end processing by the HerA-NurA complex, we analysed its 3D-architecture by SAXS. SAXS analysis of the HerA-NurA complex resulted in the generation of a molecular envelope that is compatible with the predicted co-axial arrangement of the individual HerA and NurA components (see Figure 8A). Closer inspection of the envelope confirmed a pore-like cavity in the density, permitting unambiguous docking of the hexameric HerA crystal structure (see Figure 8B). Combining this observation with knowledge of the reciprocal hydrophobic interaction surfaces on HerA and NurA enabled subsequent docking of the dimeric NurA crystal structure (16) into the remaining density. SAXS data processing and model generation steps are summarised in Figures 8C-F, and full details of the experimental procedures and data analyses are provided in the Materials and Methods.


Structure of the hexameric HerA ATPase reveals a mechanism of translocation-coupled DNA-end processing in archaea.

Rzechorzek NJ, Blackwood JK, Bray SM, Maman JD, Pellegrini L, Robinson NP - Nat Commun (2014)

SAXS analysis of the HerA-NurA complex(A) and (B) Lateral and axial views respectively of the molecular envelope generated by ab initio SAXS modelling, with crystallographic models of HerA (PDB code: 4D2I) and NurA (PDB code: 2YGK) manually docked in it. The axial view is clipped at the level of the ATPase domains of the HerA hexamer, to visualise the pore-like cavity present in the SAXS envelope, which defines the position of HerA within the map. (C) Experimental SAXS curve for the HerA-NurA complex, in the momentum transfer range 0.006<q<0.156 used for analysis (q = 4π sinθ/λ, where 2θ is the scattering angle and λ is the wavelength). (D) Guinier plot for the low-q region of the SAXS data. Linear agreement of the Guinier equation (red line) with the experimental data (blue crosses) is confirmed by low residuals (red dots) within the boundaries indicated by the arrow, enabling estimation of the radius of gyration, Rg. (E) Inter-atomic pairwise distribution function, P(r), calculated by GNOM. Data were automatically truncated to qmax≈0.16 according to the 8/Rg limit rule for ab initio modelling by DAMMIF. The P(r) function smoothly approaches zero at Dmax=158.1 Å. (F) Theoretical scattering curve (red line) for 1 of 20 ab initio models generated by DAMMIF, fitted to the experimental data (green crosses). All 20 models were superimposed, averaged and filtered by DAMAVER to generate the final model used for envelope generation in (A) and (B). The mean normalized spatial discrepancy (NSD) for the 20 aligned models was 0.619±0.048, and one model was omitted from averaging as an outlier.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376295&req=5

Figure 8: SAXS analysis of the HerA-NurA complex(A) and (B) Lateral and axial views respectively of the molecular envelope generated by ab initio SAXS modelling, with crystallographic models of HerA (PDB code: 4D2I) and NurA (PDB code: 2YGK) manually docked in it. The axial view is clipped at the level of the ATPase domains of the HerA hexamer, to visualise the pore-like cavity present in the SAXS envelope, which defines the position of HerA within the map. (C) Experimental SAXS curve for the HerA-NurA complex, in the momentum transfer range 0.006<q<0.156 used for analysis (q = 4π sinθ/λ, where 2θ is the scattering angle and λ is the wavelength). (D) Guinier plot for the low-q region of the SAXS data. Linear agreement of the Guinier equation (red line) with the experimental data (blue crosses) is confirmed by low residuals (red dots) within the boundaries indicated by the arrow, enabling estimation of the radius of gyration, Rg. (E) Inter-atomic pairwise distribution function, P(r), calculated by GNOM. Data were automatically truncated to qmax≈0.16 according to the 8/Rg limit rule for ab initio modelling by DAMMIF. The P(r) function smoothly approaches zero at Dmax=158.1 Å. (F) Theoretical scattering curve (red line) for 1 of 20 ab initio models generated by DAMMIF, fitted to the experimental data (green crosses). All 20 models were superimposed, averaged and filtered by DAMAVER to generate the final model used for envelope generation in (A) and (B). The mean normalized spatial discrepancy (NSD) for the 20 aligned models was 0.619±0.048, and one model was omitted from averaging as an outlier.
Mentions: The structure-based mutagenesis data presented here are in agreement with the coaxial arrangement for the HerA-NurA complex predicted in our previously published study (16), where the NurA dimer docks on top of the HAS-barrel face of the HerA hexamer. We note that the exposed hydrophobic patches on the surface of NurA (16) and HerA, identified by mutagenesis, appear suitably aligned to mediate this interaction (Supplementary Fig. 7). To provide further insight into the mechanism of DNA-end processing by the HerA-NurA complex, we analysed its 3D-architecture by SAXS. SAXS analysis of the HerA-NurA complex resulted in the generation of a molecular envelope that is compatible with the predicted co-axial arrangement of the individual HerA and NurA components (see Figure 8A). Closer inspection of the envelope confirmed a pore-like cavity in the density, permitting unambiguous docking of the hexameric HerA crystal structure (see Figure 8B). Combining this observation with knowledge of the reciprocal hydrophobic interaction surfaces on HerA and NurA enabled subsequent docking of the dimeric NurA crystal structure (16) into the remaining density. SAXS data processing and model generation steps are summarised in Figures 8C-F, and full details of the experimental procedures and data analyses are provided in the Materials and Methods.

Bottom Line: HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion.This system differs substantially from the bacterial end-resection paradigms.Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, UK.

ABSTRACT
The HerA ATPase cooperates with the NurA nuclease and the Mre11-Rad50 complex for the repair of double-strand DNA breaks in thermophilic archaea. Here we extend our structural knowledge of this minimal end-resection apparatus by presenting the first crystal structure of hexameric HerA. The full-length structure visualizes at atomic resolution the N-terminal HerA-ATP synthase domain and a conserved C-terminal extension, which acts as a physical brace between adjacent protomers. The brace also interacts in trans with nucleotide-binding residues of the neighbouring subunit. Our observations support a model in which the coaxial interaction of the HerA ring with the toroidal NurA dimer generates a continuous channel traversing the complex. HerA-driven translocation would propel the DNA towards the narrow annulus of NurA, leading to duplex melting and nucleolytic digestion. This system differs substantially from the bacterial end-resection paradigms. Our findings suggest a novel mode of DNA-end processing by this integrated archaeal helicase-nuclease machine.

Show MeSH
Related in: MedlinePlus