Importance of a single base pair for discrimination between intron-containing and intronless alleles by endonuclease I-Bmol

Homing endonucleases initiate mobility of their host group I introns by binding to and cleaving lengthy recognition sequences that are typically centered on the intron insertion site (IS) of intronless alleles [1, 2]. Because the intron interrupts the endonucleases' recognition sequence, intron-containing alleles are immune to cleavage by their own endonuclease [3]. I-TevI and I-Bmol are related GIY-YIG endonucleases that bind a homologous stretch of thymidylate synthase (TS)encoding DNA but use different strategies to distinguish intronless from intron-containing substrates [4-8]. I-Tevl discriminates between substrates at the level of DNA binding, as its recognition sequence is centered on the intron IS [5-7]. I-Bmol, in contrast, possesses a very asymmetric recognition sequence with respect to the intron IS, binds both intron-containing and intronless TS-encoding substrates, but efficiently cleaves only intronless substrate [8]. Here, we show that I-Bmol is extremely tolerant of multiple substitutions around its cleavage sites and has a low specific activity. However, a single G-C base pair, at position -2 of a 39-base pair recognition sequence, is a major determinant for cleavage efficiency and distinguishes intronless from intron-containing alleles. Strikingly, this G-C base pair is universally conserved in phylogenetically diverse TS-coding sequences; this finding suggests that I-Bmol has evolved exquisite cleavage requirements to maximize the potential to spread to variant intronless alleles, while minimizing cleavage at its own intron-containing allele.