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

• Published in: PloS one 2012-08, Vol.7 (8), p.e43738-e43738
• Main Authors: Kong, Shuanglei, Liu, Xinyao, Fu, Liwen, Yu, Xiangchun, An, Chengcai
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Analysis; Bacteria; Bacteriology; Bacteriophages; Bacteriophages - enzymology; Bacteriophages - genetics; Base Sequence; Biology; Calcium; Cyanobacteria; Cyanobacteria - virology; Deletion mutant; Deoxyribonuclease I - chemistry; Deoxyribonuclease I - genetics; Deoxyribonuclease I - metabolism; Deoxyribonucleic acid; DNA; DNA Cleavage; DNA polymerase; DNA polymerases; DNA, Viral - genetics; DNA, Viral - metabolism; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; E coli; Endonuclease; Escherichia coli - genetics; Escherichia coli - growth & development; Evolution, Molecular; Genes; Genetic aspects; Genomes; Homing; Homing endonuclease; Insertion; Introns - genetics; Joints; Laboratories; Life sciences; Magnesium; Molecular Sequence Data; Mutants; Nicking endonuclease; Nucleases; Nucleotide sequence; Phages; Phylogeny; Physiological aspects; Proteins; RNA Splicing; Splicing; Target recognition; Transcription
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0043738

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.