title: Chilo iridescent virus (CIV) ORF 012L encodes a protein with both exonuclease and endonuclease functions.
abstract: Chilo iridescent virus (CIV) is the type member of the genus Iridovirus within the family Iridoviridae. The virions of CIV contain a single linear dsDNA molecule that is circularly permuted and terminally redundant. The genome of CIV contains an open reading frame (ORF 012L) encoding a protein homologous to exonuclease II of Schizosaccharomyces pombe. In this study, we focused on the characterization of CIV ORF 012L. The target ORF was cloned into the pET28a vector, expressed in E. coli strain BL21 (DE3) pLysS with an N-terminal His tag and purified to homogeneity by using Ni-NTA affinity chromatography. Biochemical characterization of the purified CIV 012L confirmed that this viral protein is a functional 5'-3' exonuclease that digests 3'-biotin-labelled oligonucleotides and linear double-stranded DNA (dsDNA) molecules from their 5' termini in a highly processive manner. CIV 012L also has a potent endonuclease activity on dsDNA in vitro. In addition, CIV 012L converted supercoiled plasmid DNA (replicative form I, RFI) into the open circular form (RFII) and then open circular form into linear form (RFIII). Endonuclease activity of CIV 012L was optimal in the presence of 10 mM Mg(2+) or 30 mM Mn(2+) ions and at 150 mM NaCl or KCl salt concentrations. The highest endonuclease activity was obtained at pH 8, and it reached a maximum at 55 °C. The CIV 012L protein showed deficiencies for both double- and single-stranded RNAs.
authors: Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey.|Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University, 53100, Rize, Turkey.
authors: Yesim Akturk
authors: YA
authors: Dizman
authors: Department of Molecular Biology and Genetic, Faculty of Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors: Hacer
authors: H
authors: Muratoglu
authors: Department of Biology, Faculty of Arts and Sciences, Recep Tayyip Erdoğan University, 53100, Rize, Turkey.
authors: Cemal
authors: C
authors: Sandalli
authors: Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey. remziye@ktu.edu.tr.
authors: Remziye
authors: R
authors: Nalcacioglu
authors: Department of Biology, Faculty of Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey.
authors: Zihni
authors: Z
authors: Demirbag
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020536:Enzyme Activators / Q000032:analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D017994:Iridovirus / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D004270:DNA, Circular; D020536:Enzyme Activators; D011994:Recombinant Proteins; D014764:Viral Proteins; D004247:DNA; D004720:Endonucleases; D005092:Exonucleases