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Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease

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ABSTRACT

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ββα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.

No MeSH data available.


Effect of various Mn2+ concentrations on the DNA nicking of the wild type and mutant GVE2 HNHEs.(A) DNA nicking of the wild type GVE2 HNHE. (B) DNA nicking of the H93A mutant. (C) DNA nicking of the N109A mutant. (D) DNA nicking assays of the H119A mutant. DNA nicking reactions were performed at various reaction Mn2+ concentrations ranging from 0.002 to 10 mM by using pET-30a DNA as the substrate at 60 °C for 15 min. CK1: the reaction without the enzyme. CK2: the reaction without Mn2+. OC: open circular DNA; L: Linear DNA; CCC: covalently closed circular DNA.
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f6: Effect of various Mn2+ concentrations on the DNA nicking of the wild type and mutant GVE2 HNHEs.(A) DNA nicking of the wild type GVE2 HNHE. (B) DNA nicking of the H93A mutant. (C) DNA nicking of the N109A mutant. (D) DNA nicking assays of the H119A mutant. DNA nicking reactions were performed at various reaction Mn2+ concentrations ranging from 0.002 to 10 mM by using pET-30a DNA as the substrate at 60 °C for 15 min. CK1: the reaction without the enzyme. CK2: the reaction without Mn2+. OC: open circular DNA; L: Linear DNA; CCC: covalently closed circular DNA.

Mentions: In our previous work, we demonstrated that Mn2+ is the optimal divalent ion for DNA nicking by GVE2 HNHE only when the reactions are performed in the presence of 0.2–2 mM Mn2+ 21. To investigate the effect of Mn2+ on the wild type and mutant GVE2 HNHEs, we performed DNA nicking reactions in the presence of a broad range of Mn2+ concentrations from 2 nM to 10 mM. We found that the wild type GVE2 HNHE cleaved the cccDNA (covalently closed circular DNA) substrate to form the ocDNA (open circular DNA) product even in the presence of very low Mn2+ (2 nM) (Fig. 6A). When Mn2+ concentrations were increased to 10 μM, only ocDNA product was created by the wild type GVE2 HNHE, suggesting that 2 nM to 10 μM Mn2+ in the DNA nicking reactions enabled the enzyme to transform the cccDNA substrate to the ocDNA product. In addition, when Mn2+ concentrations were increased to more than 50 μM, smear DNA product and small linear DNA fragments were formed in DNA nicking reaction catalyzed by the wild type GVE2 HNHE. These observations suggest various concentrations of Mn2+ can stimulate GVE2 HNHE to nick the cccDNA substrate into various DNA products.


Structural and functional characterization of deep-sea thermophilic bacteriophage GVE2 HNH endonuclease
Effect of various Mn2+ concentrations on the DNA nicking of the wild type and mutant GVE2 HNHEs.(A) DNA nicking of the wild type GVE2 HNHE. (B) DNA nicking of the H93A mutant. (C) DNA nicking of the N109A mutant. (D) DNA nicking assays of the H119A mutant. DNA nicking reactions were performed at various reaction Mn2+ concentrations ranging from 0.002 to 10 mM by using pET-30a DNA as the substrate at 60 °C for 15 min. CK1: the reaction without the enzyme. CK2: the reaction without Mn2+. OC: open circular DNA; L: Linear DNA; CCC: covalently closed circular DNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304195&req=5

f6: Effect of various Mn2+ concentrations on the DNA nicking of the wild type and mutant GVE2 HNHEs.(A) DNA nicking of the wild type GVE2 HNHE. (B) DNA nicking of the H93A mutant. (C) DNA nicking of the N109A mutant. (D) DNA nicking assays of the H119A mutant. DNA nicking reactions were performed at various reaction Mn2+ concentrations ranging from 0.002 to 10 mM by using pET-30a DNA as the substrate at 60 °C for 15 min. CK1: the reaction without the enzyme. CK2: the reaction without Mn2+. OC: open circular DNA; L: Linear DNA; CCC: covalently closed circular DNA.
Mentions: In our previous work, we demonstrated that Mn2+ is the optimal divalent ion for DNA nicking by GVE2 HNHE only when the reactions are performed in the presence of 0.2–2 mM Mn2+ 21. To investigate the effect of Mn2+ on the wild type and mutant GVE2 HNHEs, we performed DNA nicking reactions in the presence of a broad range of Mn2+ concentrations from 2 nM to 10 mM. We found that the wild type GVE2 HNHE cleaved the cccDNA (covalently closed circular DNA) substrate to form the ocDNA (open circular DNA) product even in the presence of very low Mn2+ (2 nM) (Fig. 6A). When Mn2+ concentrations were increased to 10 μM, only ocDNA product was created by the wild type GVE2 HNHE, suggesting that 2 nM to 10 μM Mn2+ in the DNA nicking reactions enabled the enzyme to transform the cccDNA substrate to the ocDNA product. In addition, when Mn2+ concentrations were increased to more than 50 μM, smear DNA product and small linear DNA fragments were formed in DNA nicking reaction catalyzed by the wild type GVE2 HNHE. These observations suggest various concentrations of Mn2+ can stimulate GVE2 HNHE to nick the cccDNA substrate into various DNA products.

View Article: PubMed Central - PubMed

ABSTRACT

HNH endonucleases in bacteriophages play a variety of roles in the phage lifecycle as key components of phage DNA packaging machines. The deep-sea thermophilic bacteriophage Geobacillus virus E2 (GVE2) encodes an HNH endonuclease (GVE2 HNHE). Here, the crystal structure of GVE2 HNHE is reported. This is the first structural study of a thermostable HNH endonuclease from a thermophilic bacteriophage. Structural comparison reveals that GVE2 HNHE possesses a typical ββα-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra α-helix, suggesting conservation of these enzymes among bacteriophages. Biochemical analysis suggests that the alanine substitutions of the conserved residues (H93, N109 and H118) in the HNH motif of GVE2 HNHE abolished 94%, 60% and 83% of nicking activity, respectively. Compared to the wild type enzyme, the H93A mutant displayed almost the same conformation while the N108A and H118A mutants had different conformations. In addition, the wild type enzyme was more thermostable than the mutants. In the presence of Mn2+ or Zn2+, the wild type enzyme displayed distinct DNA nicking patterns. However, high Mn2+ concentrations were needed for the N109A and H118A mutants to nick DNA while Zn2+ inactivated their nicking activity.

No MeSH data available.