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Crystal structure of the NurA-dAMP-Mn2+ complex.

Chae J, Kim YC, Cho Y - Nucleic Acids Res. (2011)

Bottom Line: The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer.The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf NurA.We provide a DNA binding and cleavage model for Pf NurA.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, South Korea.

ABSTRACT
Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf NurA. We provide a DNA binding and cleavage model for Pf NurA.

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PfNurA–ssDNA-binding model. (A) A ssDNA (blue) is modeled on PfNurA based on the TtAgo–DNA and PfNurA–dAMP–Mn2+ structures. Box: a close up view of the model showing the direction of the ssDNA, where the 3′ OH of a ribose ring points toward the open channel (entrance) and the 5′-phosphate group is directed to the narrow part of the channel. (B) Schematic drawing of the PfNurA nuclease mechanism. Mn2+ (M1) is coordinated by Asp51, Asp126 and two water molecules. Mn2+ (M2) interacts with Asp51, His411 and one water molecule. In this model, a water nucleophile (red) bound to both metal ions attacks the phosphate group and M1 stabilizes the leaving 3′ oxyanion, which allows the 5′–3′ digestion.(C) Structure of the PfNurA showing the position of some residues on the surface of the elliptical channel. Residues containing K297E/Y380F/Y403F (blue label), R323E/R435E (black label) and K415E/K419E (red label) are shown. (D) Nuclease activity assay of the three DNA-binding mutant using TP 424/423 or (E) TP 580/124. Each DNA substrate (20 nM) was incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35 and 70 nM), 5 mM MnCl2 and 1 mM ATP. In lane 2, 70 nM of PfHerA was used as a control. Abbreviations: K297E*, K297E/Y380F/Y403F; R323E*, R323E/R435E; K415E*, K415E/K419E.
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gkr999-F6: PfNurA–ssDNA-binding model. (A) A ssDNA (blue) is modeled on PfNurA based on the TtAgo–DNA and PfNurA–dAMP–Mn2+ structures. Box: a close up view of the model showing the direction of the ssDNA, where the 3′ OH of a ribose ring points toward the open channel (entrance) and the 5′-phosphate group is directed to the narrow part of the channel. (B) Schematic drawing of the PfNurA nuclease mechanism. Mn2+ (M1) is coordinated by Asp51, Asp126 and two water molecules. Mn2+ (M2) interacts with Asp51, His411 and one water molecule. In this model, a water nucleophile (red) bound to both metal ions attacks the phosphate group and M1 stabilizes the leaving 3′ oxyanion, which allows the 5′–3′ digestion.(C) Structure of the PfNurA showing the position of some residues on the surface of the elliptical channel. Residues containing K297E/Y380F/Y403F (blue label), R323E/R435E (black label) and K415E/K419E (red label) are shown. (D) Nuclease activity assay of the three DNA-binding mutant using TP 424/423 or (E) TP 580/124. Each DNA substrate (20 nM) was incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35 and 70 nM), 5 mM MnCl2 and 1 mM ATP. In lane 2, 70 nM of PfHerA was used as a control. Abbreviations: K297E*, K297E/Y380F/Y403F; R323E*, R323E/R435E; K415E*, K415E/K419E.

Mentions: Based on the Pf NurA–dAMP–Mn2+, TtAgo–DNA and EndoV–DNA crystal structures, we modeled both dsDNA and ssDNA on an active site of Pf NurA (Figure 6A and Supplementary Figure S5). DNA primarily binds to PIWI and PAZ in TtAgo, and the 3′-end is recognized by the N domain and the 5′-end is recognized by the Mid domain (38). When the PIWI domain of TtAgo is superimposed on the equivalent domain of Pf NurA, dsDNA bound to a sheet comprised of strands (S3, S2, S1, S4, S10, H13, S12 and S11), and regions, including helices H11 and H12 and strands S11 and S12 from another Pf NurA, encircled the DNA completely. However, helix H3 from one monomer and strands S1–S3 from another Pf NurA collided with the modeled dsDNA, implying that a conformational change in this region or dsDNA is necessary to accommodate the dsDNA (Supplementary Figure S5).Figure 6.


Crystal structure of the NurA-dAMP-Mn2+ complex.

Chae J, Kim YC, Cho Y - Nucleic Acids Res. (2011)

PfNurA–ssDNA-binding model. (A) A ssDNA (blue) is modeled on PfNurA based on the TtAgo–DNA and PfNurA–dAMP–Mn2+ structures. Box: a close up view of the model showing the direction of the ssDNA, where the 3′ OH of a ribose ring points toward the open channel (entrance) and the 5′-phosphate group is directed to the narrow part of the channel. (B) Schematic drawing of the PfNurA nuclease mechanism. Mn2+ (M1) is coordinated by Asp51, Asp126 and two water molecules. Mn2+ (M2) interacts with Asp51, His411 and one water molecule. In this model, a water nucleophile (red) bound to both metal ions attacks the phosphate group and M1 stabilizes the leaving 3′ oxyanion, which allows the 5′–3′ digestion.(C) Structure of the PfNurA showing the position of some residues on the surface of the elliptical channel. Residues containing K297E/Y380F/Y403F (blue label), R323E/R435E (black label) and K415E/K419E (red label) are shown. (D) Nuclease activity assay of the three DNA-binding mutant using TP 424/423 or (E) TP 580/124. Each DNA substrate (20 nM) was incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35 and 70 nM), 5 mM MnCl2 and 1 mM ATP. In lane 2, 70 nM of PfHerA was used as a control. Abbreviations: K297E*, K297E/Y380F/Y403F; R323E*, R323E/R435E; K415E*, K415E/K419E.
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gkr999-F6: PfNurA–ssDNA-binding model. (A) A ssDNA (blue) is modeled on PfNurA based on the TtAgo–DNA and PfNurA–dAMP–Mn2+ structures. Box: a close up view of the model showing the direction of the ssDNA, where the 3′ OH of a ribose ring points toward the open channel (entrance) and the 5′-phosphate group is directed to the narrow part of the channel. (B) Schematic drawing of the PfNurA nuclease mechanism. Mn2+ (M1) is coordinated by Asp51, Asp126 and two water molecules. Mn2+ (M2) interacts with Asp51, His411 and one water molecule. In this model, a water nucleophile (red) bound to both metal ions attacks the phosphate group and M1 stabilizes the leaving 3′ oxyanion, which allows the 5′–3′ digestion.(C) Structure of the PfNurA showing the position of some residues on the surface of the elliptical channel. Residues containing K297E/Y380F/Y403F (blue label), R323E/R435E (black label) and K415E/K419E (red label) are shown. (D) Nuclease activity assay of the three DNA-binding mutant using TP 424/423 or (E) TP 580/124. Each DNA substrate (20 nM) was incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35 and 70 nM), 5 mM MnCl2 and 1 mM ATP. In lane 2, 70 nM of PfHerA was used as a control. Abbreviations: K297E*, K297E/Y380F/Y403F; R323E*, R323E/R435E; K415E*, K415E/K419E.
Mentions: Based on the Pf NurA–dAMP–Mn2+, TtAgo–DNA and EndoV–DNA crystal structures, we modeled both dsDNA and ssDNA on an active site of Pf NurA (Figure 6A and Supplementary Figure S5). DNA primarily binds to PIWI and PAZ in TtAgo, and the 3′-end is recognized by the N domain and the 5′-end is recognized by the Mid domain (38). When the PIWI domain of TtAgo is superimposed on the equivalent domain of Pf NurA, dsDNA bound to a sheet comprised of strands (S3, S2, S1, S4, S10, H13, S12 and S11), and regions, including helices H11 and H12 and strands S11 and S12 from another Pf NurA, encircled the DNA completely. However, helix H3 from one monomer and strands S1–S3 from another Pf NurA collided with the modeled dsDNA, implying that a conformational change in this region or dsDNA is necessary to accommodate the dsDNA (Supplementary Figure S5).Figure 6.

Bottom Line: The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer.The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf NurA.We provide a DNA binding and cleavage model for Pf NurA.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Science, Pohang University of Science and Technology, Pohang 790-784, South Korea.

ABSTRACT
Generation of the 3' overhang is a critical event during homologous recombination (HR) repair of DNA double strand breaks. A 5'-3' nuclease, NurA, plays an important role in generating 3' single-stranded DNA during archaeal HR, together with Mre11-Rad50 and HerA. We have determined the crystal structures of apo- and dAMP-Mn(2)(+)-bound NurA from Pyrococcus furiousus (Pf NurA) to provide the basis for its cleavage mechanism. Pf NurA forms a pyramid-shaped dimer containing a large central channel on one side, which becomes narrower towards the peak of the pyramid. The structure contains a PIWI domain with high similarity to argonaute, endoV nuclease and RNase H. The two active sites, each of which contains Mn(2)(+) ion(s) and dAMP, are at the corners of the elliptical channel near the flat face of the dimer. The 3' OH group of the ribose ring is directed toward the channel entrance, explaining the 5'-3' nuclease activity of Pf NurA. We provide a DNA binding and cleavage model for Pf NurA.

Show MeSH