Dataset Information

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

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 ???-metal fold and Zn-finger motif similar to those of HNH endonucleases from other bacteriophages, apart from containing an extra ?-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.

SUBMITTER: Zhang L

PROVIDER: S-EPMC5304195 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Zhang Likui L Xu Dandan D Huang Yanchao Y Zhu Xinyuan X Rui Mianwen M Wan Ting T Zheng Xin X Shen Yulong Y Chen Xiangdong X Ma Kesen K Gong Yong Y

Scientific reports 20170213

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 moti ...[more]

PMID: 28211904

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Project description:To investigate the transcriptional profile of GVE2 genes, the viral genes were identified by DNA microarray with Cy5- or Cy3-dUTP-labeled cDNAs prepared from uninfected and GVE2-infected Geobacillus sp. E263 at 4 h p.i.. After hybridization with the Cy3-dUTP-labeled cDNAs from GVE2-infected Geobacillus sp. E263 at 4 h p.i., Cy5-dUTP-labeled cDNAs from uninfected Geobacillus sp. E263 as well as Cy3- dUTP-labeled yeast cDNAs and Hex DNA, many spots produced positive signals significantly above the background, while no signal appeared for the Cy5-dUTP-labeled cDNAs from uninfected Geobacillus sp. E263., indicating that the positive signals represented the GVE2 gene transcripts detectable by DNA microarray. The DNA fragments, detected to be positive in the reverse transcripts at 4 h p.i., contained 74.2% of the presumptive GVE2 ORFs. Keywords: Transcriptional profile of thermophilic bacteriophage at 4 h p.i. A DNA microarray containing 82 DNA fragments of the viral genome was constructed following a PCR-based microarray method. Briefly, specific primer sets were designed to amplify approximately 500-bp fragment each using viral genome as template. All PCR products showing a single band of the appropriate size by gel electrophoresis were purified, and reconstituted in TE buffer at a final concentration of about 500 Î¼g/ml for spotting in triplicates onto the silylated-glass slides (CEL Associates, Inc. USA) using a microarrayer (Smart Arrayer 48, CapitalBio). Eight DNA fragments from yeast genome and a randomly synthesized DNA fragment (Hex) were included as exogenous positive controls to normalize the microarry date. Distilled water was used as negative controls. Total RNAs were isolated from the uninfected and phage-infected Geobacillus sp. E263 cells at 4 h postinfection. The cDNAs from uninfected Geobacillus sp. E263 were labeled with Cy5 and the cDNAs from phage-infected Geobacillus sp. E263 labeled with Cy3. At the same time, the cDNAs from yeast and the Hex DNA were labeled with Cy3. The Cy5- or Cy3-dUTP-labeled cDNAs were resuspended in hybridization solution and hybridized with the microarrays for 16 to 18 h at 42Â°C. Then the microarrays were rinsed several times following the standard method. Following the washing steps, the microarrays were dried by low-speed centrifugation (500 g for 5 min), and immediately scanned using a GenePix 4000B array scanner (Axon Instruments, Inc.). Images obtained from scanning were analyzed by GenePix Pro 4.0 array analysis software (Axon Instruments, Inc.)

Dataset Information

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

Publications

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

Similar Datasets

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