I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

Mohamed Hafez; Tuhin Kumar Guha; Georg Hausner

doi:10.1016/j.funbio.2014.05.002

I-OmiI and I-OmiII: two intron-encoded homing endonucleases within the Ophiostoma minus rns gene

Fungal Biol. 2014 Aug;118(8):721-31. doi: 10.1016/j.funbio.2014.05.002. Epub 2014 May 23.

Authors

Mohamed Hafez¹, Tuhin Kumar Guha², Georg Hausner³

Affiliations

¹ Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada; Department of Botany, Faculty of Science, Suez University, Suez, Egypt.
² Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada.
³ Department of Microbiology, University of Manitoba, Winnipeg, MB R3T 2N2, Canada. Electronic address: hausnerg@cc.umanitoba.ca.

PMID: 25110134
DOI: 10.1016/j.funbio.2014.05.002

Abstract

The mitochondrial small subunit ribosomal RNA (rns) gene of the ascomycetous fungus Ophiostoma minus [strain WIN(M)371] was found to contain a group IC2 and a group IIB1 intron at positions mS569 and mS952 respectively. Both introns have open reading frames (ORFs) embedded that encode double motif LAGLIDADG homing endonucleases (I-OmiI and I-OmiII respectively). Codon-optimized versions of I-OmiI and I-OmiII were synthesized for overexpression in Escherichia coli. The in vitro characterization of I-OmiII showed that it is a functional homing endonuclease that cleaves the rns target site two nucleotides upstream (sense strand) of the intron insertion site generating 4 nucleotide 3' overhangs. The endonuclease activity of I-OmiII was tested using linear and circular substrates and cleavage activity was evaluated at various temperatures. The I-OmiI protein was expressed in E. coli, but purification was difficult, thus the endonuclease activity of this protein was tested via in vivo assays. Overall this study showed that there are many native forms of functional homing endonucleases yet to be discovered among fungal mtDNA genomes.

Keywords: DNA cutting enzymes; In vivo endonuclease assays; Mitochondrial DNA; Mobile elements; Mobile introns.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cloning, Molecular
DNA, Mitochondrial / genetics*
Endonucleases / genetics*
Escherichia coli / genetics
Gene Expression
Genes, rRNA*
Introns*
Molecular Sequence Data
Open Reading Frames
Ophiostoma / enzymology*
Ophiostoma / genetics
Sequence Analysis, DNA

Substances

DNA, Mitochondrial
Endonucleases

Associated data

GENBANK/HQ292080
GENBANK/HQ292082