title: I-PfoP3I: a novel nicking HNH homing endonuclease encoded in the group I intron of the DNA polymerase gene in Phormidium foveolarum phage Pf-WMP3.
abstract: Homing endonucleases encoded in a group I self-splicing intron in a protein-coding gene in cyanophage genomes have not been reported, apart from some free-standing homing edonucleases. In this study, a nicking DNA endonuclease, I-PfoP3I, encoded in a group IA2 intron in the DNA polymerase gene of a T7-like cyanophage Pf-WMP3, which infects the freshwater cyanobacterium Phormidium foveolarum is described. The Pf-WMP3 intron splices efficiently in vivo and self-splices in vitro simultaneously during transcription. I-PfoP3I belongs to the HNH family with an unconventional C-terminal HNH motif. I-PfoP3I nicks the intron-minus Pf-WMP3 DNA polymerase gene more efficiently than the Pf-WMP4 DNA polymerase gene that lacks any intervening sequence in vitro, indicating the variable capacity of I-PfoP3I. I-PfoP3I cleaves 4 nt upstream of the intron insertion site on the coding strand of EXON 1 on both intron-minus Pf-WMP3 and Pf-WMP4 DNA polymerase genes. Using an in vitro cleavage assay and scanning deletion mutants of the intronless target site, the minimal recognition site was determined to be a 14 bp region downstream of the cut site. I-PfoP3I requires Mg(2+), Ca(2+) or Mn(2+) for nicking activity. Phylogenetic analysis suggests that the intron and homing endonuclease gene elements might be inserted in Pf-WMP3 genome individually after differentiation from Pf-WMP4. To our knowledge, this is the first report of the presence of a group I self-splicing intron encoding a functional homing endonuclease in a protein-coding gene in a cyanophage genome.
authors.affiliation: State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
authors.forename: Shuanglei
authors.initials: S
authors.lastname: Kong
authors.forename: Xinyao
authors.initials: X
authors.lastname: Liu
authors.forename: Liwen
authors.initials: L
authors.lastname: Fu
authors.forename: Xiangchun
authors.initials: X
authors.lastname: Yu
authors.forename: Chengcai
authors.initials: C
authors.lastname: An
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D000458:Cyanobacteria / Q000821:virology*; D053837:DNA Cleavage; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D019143:Evolution, Molecular; D007438:Introns / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012326:RNA Splicing
chemical_list: D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase; D003850:Deoxyribonuclease I