Group II Intron Homing Endonucleases

Thus far, DNA target sites have been defined experimentally for the yeast introns coxl-ll (Yang et al. 1998) and coxl-12 (Guo et al. 1997), the L. lactis Ll.LtrB intron (Guo et al. 2000; Mohr et al. 2000; Perutka et al. 2004), and the S. meliloti Rmlntl intron (Jiménez-Zurdo et al. 2003; Fig. 4). All four introns use the same basic mechanism of DNA target site recognition involving both the recognition of specific nucleotide residues by the IEP and base pairing of the

Fig. 4. Group II intron DNA target site interactions. Shown are target sites for the L. lactis Ll.LtrB intron (Mohr et al. 2000; Singh and Lambowitz 2001; Perutka et al. 2004), S. cerevi-siae coxl- II (Yang et al. 1998), S. cerevisiae coxl-12 (Guo et al. 1997), and the S. meliloti Rmlntl intron (Jiménez-Zurdo et al. 2003). Critical positions that affect intron mobility reactions are shaded (see references for details). The in-tron-insertion site (IS) is in the top strand at the exon junction (vertical line); the bottom-strand cleavage site (CS) is indicated by an arrowhead. Intron RNA sequences base pairing with the DNA target site are shown above. The IEP is shown only for the Ll.LtrB intron intron RNA to the 5'-exon sequences IBS2 and IBS1, and the 3'-exon sequence 6' (group IIA introns) or IBS3 (group IIB introns). For each of the base-pairing interactions, mutations in the DNA target site that inhibit reverse splicing in vitro or intron mobility in vivo could be rescued by compensatory mutations in the intron RNA. Notably, analysis of reverse splicing of a group II intron into RNA substrates showed that single-base mismatches affect the kcat for reverse splicing as well as Kd and thus have much stronger inhibitory effects than are expected from decreased binding affinity alone (Xiang et al. 1998). This feature likely contributes to the very high target specificity for intron insertion.

For the yeast coxl-ll and -12 and LLLtrB introns, which contain C-termi-nal D and En domains, the IEP recognizes specific sequences both upstream and downstream of the IBS and 6 sequences. For all three introns, mutations in key nucleotide residues recognized by the IEP in the distal 5'-exon region inhibit both reverse splicing and second-strand cleavage, while mutations at 3'-exon positions recognized by the IEP inhibit only second-strand cleavage. Rmlntl, whose IEP lacks the En domain as well as all or part of domain D, uses the same basic mode of DNA target site recognition. However, recognition by the IEP appears more limited, with mutations at only two positions (-15 and +4) strongly inhibiting mobility (Jiménez-Zurdo et al. 2003). A caveat is that the deletion of 5'-exon sequences between positions -20 and -16 decreased the homing efficiency by >95%, suggesting that there could also be significant recognition determinants in this region (Jiménez-Zurdo et al. 2003). Rmlntl does not carry out second-strand cleavage, so the 3'-exon mutations must inhibit mobility either by affecting the initial DNA target site recognition or reverse splicing steps. The latter situation differs from that for the yeast and lactoccal introns where 3'-exon mutations inhibit only second-strand cleavage (see above).

Bacterial class C introns, which also lack the En domain, are found inserted downstream of palindromic rho-independent transcription terminators at sites that can potentially form only the EBS1/IBS1 pairing with the intron RNA (Granlund et al. 2001; Yeo et al. 2001; Dai and Zimmerly 2002). These introns may use a variation of the DNA target site recognition mechanism in which the IEP has adapted to recognize a specific higher-order DNA structure. Indeed, the formation of this palindromic structure may be favored in transiently single-stranded DNA regions at replication forks, thus targeting the introns to regions where they can use nascent lagging strands to prime reverse transcription (see above; Lambowitz and Zimmerly 2004).

Comparison of different target sites shows that even closely related group II introns, like yeast coxl-ll and -12, have different target specificities both in the region recognized by base pairing of the intron RNA and in the flanking regions recognized by the IEP. These findings suggest that the IEPs can readi ly evolve or adapt to recognize different target site sequences, enabling group II introns to establish themselves at new sites.

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