Importance of a Single Base Pair for Discrimination between Intron-Containing and Intronless Alleles by Endonuclease I-BmoI

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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-BmoI 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-TevI discriminates between substrates at the level of DNA binding, as its recognition sequence is centered on the intron IS [5–7]. I-BmoI, 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-BmoI 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-BmoI has evolved exquisite cleavage requirements to maximize the potential to spread to variant intronless alleles, while minimizing cleavage at its own intron-containing allele.