Studies of the homing endonuclease I-CreII with respect to the roles of the GIY-YIG and H-N-H domains
Homing endonucleases (HEs) typically have one of four types of catalytic domains (LAGLIDADG, GIY-YIG, H-N-H, His-Cys), and a DNA-binding region(s) that provides specificity. I-CreII, which is encoded by the psbA4 intron from Chlamydomonas reinhardtii, is unusual in containing two catalytic motifs: H-N-H and GIY-YIG. A previous study showed that I-CreII cleavage leaves 2-nt 3' OH overhangs similar to GIYYIG endonucleases, but that it also has a flexible metal requirement like H-N-H enzymes. Also, alanine substitution of several conserved residues in the GIY-YIG motif and two in the H-N-H motif did not produce a clear catalytic mutant, although some variants had strongly reduced DNA binding. Thus, in order to identify the catalytic motif, I substituted additional amino acids in both domains with alanine, and identified three histidines in the H-N-H motif that are likely to be involved in catalysis. To gain insight into how I-CreII interacts with its ~30-bp homing-site DNA, three types of DNA protection analysis were performed. Hydroxyl-radical footprinting, which reveals regions of tight DNA binding, indicated that I-CreII binds strongly to a region downstream of the cleavage and intron-insertion sites, corresponding to bp 2-10 of exon 5. There was also partial protection around the cleavage site, but only on the top strand, which is consistent with the enzyme's tendency to cleave this strand first. DNase I protection, which can reveal less closely-bound regions of target DNA, gave a larger footprint than hydroxyl-radical protection, with the additional region lying upstream of the cleavage site. These results also suggest that DNA backbone-binding downstream of the cleavage site involves sugars and phosphates, whereas upstream it is mainly with phosphates. DMS protection, which probes guanines on the N-7 position in the major groove, did not provide any evidence of major groove binding (at least not through guanines). DNase I protection could also be performed on the I-CreII variants that had reduced DNA affinity. The N161A variant was instructive in that it showed reduced protection of a T-A bp very close to the cleavage site, providing support for a catalytic role for the H-N-H motif and a possible constraint for modeling. Of the GIY-YIG motif variants, the footprint of the G231E/K245A variant was distinctly useful in that it was preferentially effected downstream of the cleavage site. This result suggested the H-N-H and GIY-YIG motifs are co-linear with their targets in the homing site. Structural modeling of the GIY-YIG domain of I-CreII using the solved I-TevI domain as template provided evidence for a unique insertion in the I-CreII structure that disrupted a catalytic α-helix; the insertion is predicted to be a positively charged, hairpinlike loop anchored by two antiparallel β-strands. I propose that this insertion can explain the evolutionary conversion of this catalytic endonuclease domain into a DNA-binding domain. These findings should also help to understand other dual-motif H-N-H/GIY-YIG endonucleases, such as I-CmoeI.