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 Mg2+, Ca2+ or Mn2+ 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.
Highlights
Group I introns are self-splicing RNA sequences that are inserted into genes of a diverse range of bacteriophages of gramnegative bacteria, gram-positive bacteria and cyanobacteria
The intervening sequence (IVS) was shown to be removable from the Pf-WMP3 DNA polymerase (DNAP) precursor mRNA that was being translated in E. coli (BL21/pETP3DNAP[+int], data not shown)
A group I Intron in the Genome of Cyanophage Pf-WMP3 Self-splicing group I introns are rarexly found in T7-like phages
Summary
Group I introns are self-splicing RNA sequences that are inserted into genes of a diverse range of bacteriophages of gramnegative bacteria, gram-positive bacteria and cyanobacteria. Many group I introns contain reading frames encoding a homing endonuclease gene (HEG) which is described as selfish genetic element [8]. Homing endonucleases (HEases) cleave single (nick) or double (DSB) strands at or close to the intron insertion site (IIS), generating strand breaks in homologous alleles that lack the intervening sequence (IVS). The strand breaks are repaired by homologous recombination using the allele that contains the HEG as a template [9]. Different from typical intron-encoded HEases, I-HmuI and I-HmuII can cleave both intron-plus and intronless versions of their cognate genes
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
