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A homing endonuclease‐rps3 gene fusion in Ophiostoma novo‐ulmi (Dutch Elm disease fungus)

In O. novo‐ulmi a homing endonuclease gene (HEG) is sometimes located within the mitochondrial (mt) rnl gene, rps3. Homing endonucleases (HE) are DNA cutting enzymes that cleave at asymmetric recognition sites (12‐40bp) and HEGs are mobile elements that can be free standing or encoded within group I or group II introns. Here we characterize the LAGLIDADG type homing endonuclease: I‐Onu1, that exists as part of a fusion protein, as the HEG inserted in‐frame into the C‐terminus of the mt‐rps3 gene. Within the filamentous ascomycetes fungi rps3 is encoded by a group I intron located within the mt‐rnl gene. Sequencing analysis of rps3 HEG (−) and HEG (+) strains suggests the presence of 4bp direct repeats (GAAT) flanking the HEG insertion sites. This is unexpected as exonucleolytic activity associated with HEG mobility is thought to remove any overhangs generated by the HE. In vitro endonuclease assays using plasmid DNA that contains the rps3 HEG (−) allele (pRPS3) confirmed HE activity. The cleavage sites were mapped using P32 5′end‐labeled 201bp PCR fragment (with predicted HE target site) derived from rps3. The results showed that the HE cuts at GAAT generating a 3′ GAAT overhang confirming the sequence data analysis. Overall this endonuclease is unique as it inserts into an essential gene and generates 4bp direct repeats usually not observed in HEG mobility events. In general, the rare cutting HEases with characterized target/recognition and cleavage sites with 3′ overhangs are useful in genetic engineering and genomics.