Related homing endonucleases I-Bmol and I-Tevl use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease. I-Bmol, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-Bmol binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-Bmol is encoded by a group I intron that interrupts the thymidylate synthase CTS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene ltd) of Escherichia coli phage T4. I-Tevl, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-Bmol, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-Tevl and I-Bmol bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.