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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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Biochemical properties of PfNurA and PfHerA (A) Comparison of nuclease activities of PfNurA and dimeric interface mutants. TP 424/423 (lanes 1–7) or TP 580/124 (lanes 8–14) DNA substrates (20 nM) were incubated for 120 min at 65°C with increasing amounts of PfNurA (350 and 1750 nM) and 5 mM MnCl2. 27-C (Δ1–26) and R11A/I12E/S60Y (R11A*) are the PfNurA dimeric interface mutant proteins. Reaction mixtures were analyzed on 15% denaturing polyacrylamide gels containing 7 M urea in TBE buffer. Schematic diagrams of each substrate with a 32P-labeled 5′-end (asterisk) and cleavage sites (arrows) are shown on the top. Five nucleotides in one 3′-end are connected through phosphorothioate bonds, which are shown as ‘SSSSS’. ssDNA markers are indicated. (B) Nuclease activities of PfNurA in the presence of PfHerA. DNA substrates (20 nM) were incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35, 70 and 115 nM), 5 mM MnCl2 and 1 mM ATP. Lanes 2 and 8 contain 115 nM PfHerA.
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gkr999-F1: Biochemical properties of PfNurA and PfHerA (A) Comparison of nuclease activities of PfNurA and dimeric interface mutants. TP 424/423 (lanes 1–7) or TP 580/124 (lanes 8–14) DNA substrates (20 nM) were incubated for 120 min at 65°C with increasing amounts of PfNurA (350 and 1750 nM) and 5 mM MnCl2. 27-C (Δ1–26) and R11A/I12E/S60Y (R11A*) are the PfNurA dimeric interface mutant proteins. Reaction mixtures were analyzed on 15% denaturing polyacrylamide gels containing 7 M urea in TBE buffer. Schematic diagrams of each substrate with a 32P-labeled 5′-end (asterisk) and cleavage sites (arrows) are shown on the top. Five nucleotides in one 3′-end are connected through phosphorothioate bonds, which are shown as ‘SSSSS’. ssDNA markers are indicated. (B) Nuclease activities of PfNurA in the presence of PfHerA. DNA substrates (20 nM) were incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35, 70 and 115 nM), 5 mM MnCl2 and 1 mM ATP. Lanes 2 and 8 contain 115 nM PfHerA.

Mentions: Previous studies have reported that NurA from Sulfolobous acidocaldarius (SaNurA) exhibits 5–3′ exonuclease activity which produced ~5 nt from the 5′-end of a substrate (16). Pf NurA also displays exonuclease activity (15). However, it is unclear if Pf NurA produces any specific cleavage products like SaNurA. To understand if the products of the archaeal NurA nucleases are conserved, we further examined the nuclease activities of Pf NurA using a 5′[32P]-labeled dsDNA oligonucleotide substrate with a 16-nt 3′-overhang (TP424/423) and a 50-nt duplex DNA containing five phosphorothioate bonds at the 3′-end of the top strand (TP580/124). Pf NurA (350 nM) in the presence of Mn2+ exhibited weak endonuclease activity on 20 nM of each substrate, and cleaved primarily ~10 nt from the 5′-end (Figure 1A, lanes 2 and 9). In the presence of a higher amount of Pf NurA (1750 nM), smaller amounts of 6- and 8-nt products were generated from the 5′-end of these substrates (Figure 1A, lanes 3 and 10).Figure 1.


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

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

Biochemical properties of PfNurA and PfHerA (A) Comparison of nuclease activities of PfNurA and dimeric interface mutants. TP 424/423 (lanes 1–7) or TP 580/124 (lanes 8–14) DNA substrates (20 nM) were incubated for 120 min at 65°C with increasing amounts of PfNurA (350 and 1750 nM) and 5 mM MnCl2. 27-C (Δ1–26) and R11A/I12E/S60Y (R11A*) are the PfNurA dimeric interface mutant proteins. Reaction mixtures were analyzed on 15% denaturing polyacrylamide gels containing 7 M urea in TBE buffer. Schematic diagrams of each substrate with a 32P-labeled 5′-end (asterisk) and cleavage sites (arrows) are shown on the top. Five nucleotides in one 3′-end are connected through phosphorothioate bonds, which are shown as ‘SSSSS’. ssDNA markers are indicated. (B) Nuclease activities of PfNurA in the presence of PfHerA. DNA substrates (20 nM) were incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35, 70 and 115 nM), 5 mM MnCl2 and 1 mM ATP. Lanes 2 and 8 contain 115 nM PfHerA.
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gkr999-F1: Biochemical properties of PfNurA and PfHerA (A) Comparison of nuclease activities of PfNurA and dimeric interface mutants. TP 424/423 (lanes 1–7) or TP 580/124 (lanes 8–14) DNA substrates (20 nM) were incubated for 120 min at 65°C with increasing amounts of PfNurA (350 and 1750 nM) and 5 mM MnCl2. 27-C (Δ1–26) and R11A/I12E/S60Y (R11A*) are the PfNurA dimeric interface mutant proteins. Reaction mixtures were analyzed on 15% denaturing polyacrylamide gels containing 7 M urea in TBE buffer. Schematic diagrams of each substrate with a 32P-labeled 5′-end (asterisk) and cleavage sites (arrows) are shown on the top. Five nucleotides in one 3′-end are connected through phosphorothioate bonds, which are shown as ‘SSSSS’. ssDNA markers are indicated. (B) Nuclease activities of PfNurA in the presence of PfHerA. DNA substrates (20 nM) were incubated for 120 min at 65°C with 350 nM of PfNurA, increasing amounts of PfHerA (35, 70 and 115 nM), 5 mM MnCl2 and 1 mM ATP. Lanes 2 and 8 contain 115 nM PfHerA.
Mentions: Previous studies have reported that NurA from Sulfolobous acidocaldarius (SaNurA) exhibits 5–3′ exonuclease activity which produced ~5 nt from the 5′-end of a substrate (16). Pf NurA also displays exonuclease activity (15). However, it is unclear if Pf NurA produces any specific cleavage products like SaNurA. To understand if the products of the archaeal NurA nucleases are conserved, we further examined the nuclease activities of Pf NurA using a 5′[32P]-labeled dsDNA oligonucleotide substrate with a 16-nt 3′-overhang (TP424/423) and a 50-nt duplex DNA containing five phosphorothioate bonds at the 3′-end of the top strand (TP580/124). Pf NurA (350 nM) in the presence of Mn2+ exhibited weak endonuclease activity on 20 nM of each substrate, and cleaved primarily ~10 nt from the 5′-end (Figure 1A, lanes 2 and 9). In the presence of a higher amount of Pf NurA (1750 nM), smaller amounts of 6- and 8-nt products were generated from the 5′-end of these substrates (Figure 1A, lanes 3 and 10).Figure 1.

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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