How Intertwined Are the DNA and RNA Activities in Maturases

The fact that bifunctional LAGLIDADG DNA endonucleases/RNA maturases exist raises the question of whether or not such proteins use a single binding site to interact with their nucleic acid ligands. Indeed, the similarity between respective DNA-binding sites and the RNA structure around the 5' and 3' SS (P1/P10 pseudoknot; Fig. 2b) has fueled speculation that such proteins may use analogous interactions to perform both functions (reviewed in Lam-bowitz et al. 1999). However, in vitro experiments with the A.n. COB I-An-il maturase showed that disruption of the COB pre-mRNA's P1/P10 by deletion of PI or the 3' exon has only modest effects on I-Anil binding (Solem et al. 2002). These observations suggest that protein interactions in and around this region, if they exist, contribute little to binding affinity and that maturases recognize their introns in a more complex manner than had originally been speculated.

Structural, genetic and biochemical evidence have been brought to bear on the question of how bifunctional endonuclease/maturases interact with their ligands. A cocrystal of I-Anil endonuclease/maturase complexed with its DNA substrate was recently solved (Bolduc et al. 2003; Fig. lc). Overall, the structure of I-Anil is similar to those of other dedicated LAGLIDADG endo-

nucleases that have also been crystallized (Chevalier and Stoddard 2001). The two LAGLIDADG motifs have a-helical conformation and pack against one another to form a central axis. Perpendicular to each helix is a curved, four-stranded, antiparallel P-sheet. The two P-sheets, one each from the N- and C-terminal domains, form an extended groove that binds to the DNA substrate making multiple specific base and non-specific phosphate contacts along 19 nucleotides (Fig. lc). Remarkably, I-Anil shows no alterations or additions to the basic homing endonuclease framework despite its "extra" function.

Several lines of evidence suggest that maturases bind their cognate in-tron RNA using a distinct surface from their DNA-binding site. First, mutations in yeast bifunctional maturases have been recovered that differentially affect maturase and endonuclease functions. The homing endonuclease I-Scell, encoded by aI4a intron, functions as a latent maturase but still retains its homing activity (see above). Mutations in the third or ninth position of the first LAGLIDADG motif abolish endonuclease activity, while mutations in the same positions of the second motif only affect maturase activity (Henke et al. 1995). In addition, mutations in the acidic residue at the eighth position of the first and second LAGLIDADG motifs specifically affect DNA endonuclease activity in the endonuclease/maturases I-Scal and I-Anil (Szczepanek et al. 2000; Chatterjee et al. 2003). Second, if maturases use the endonuclease extended groove to bind intron RNA, competition experiments between DNA substrate and intron RNA would show that binding of one substrate negates the binding of the other. Indeed, in the case of I-Anil, the A.n. COB intron RNA is an effective competitor for DNA cleavage and binding by I-Anil (Chatterjee et al. 2003; Geese et al. 2003). Surprisingly, however, the presence of the DNA substrate showed no observable effect on splicing of the pre-mR-NA (Chatterjee et al. 2003; Geese et al. 2003). In addition, the presence of the DNA target site did not affect the binding of the full-length COB intron RNA or a mutant RNA that binds the protein ~30-fold less tightly than the competitor DNA (Chatterjee et al. 2003). Taken together, these observations suggest that maturases have functionally distinct RNA-binding sites. Presumably, I-Anil binding to the COB intron causes a conformational change in the protein that negatively affects DNA substrate binding (Chatterjee et al. 2003).

Clues to the location of the intron RNA-binding site have come from mutational analysis of the I-Anil maturase. Alignment of I-Anil with two of its closest maturase relatives (encoded by the S. cerevisiae bI3 and V. inaequalis bI5 introns) revealed conserved basic residues that fall on one side of I-An-il not in contact with the DNA (Bolduc et al. 2003). A single Arg to Glu substitution on this surface of the C-terminus of I-Anil reduced intron binding and splicing ~10-fold while having no effects on DNA substrate hydrolysis or affinity (Bolduc et al. 2003). Other maturase-specific mutations in the C-ter-minus have also been identified (Kwon and Waring, in prep.). Significantly, a C-terminal fragment of I-Anil that begins at the second LAGLIDADG motif does not cleave substrate DNA, as expected, but does facilitate splicing of COB intron almost as efficiently as the full-length protein. The corresponding N-terminal fragment, beginning at the first and ending at the second motif, has no splicing activity. Other experiments show that the C-terminal protein interacts with the COB intron in a manner directly analogous to the full-length protein (Downing et al. 2005). These observations are consistent with "domain" swapping experiments with the S. cerevisiae bI4 maturase and wildtype aI4a endonuclease that share extensive amino acid conservation (Dela-hodde et al. 1989). The analysis of hybrid proteins in vivo showed that while an N-terminal aI4a/C-terminal bI4 protein has robust RNA maturase activity, an N-terminal bI4/C-terminal aI4a protein has only weak maturase activity (Goguel et al. 1992). Taken together, these data show that splicing function is derived, for the most part, from the C-terminus of maturases on a surface opposite the DNA-binding site. These observations support a model in which homing endonucleases have developed maturase function by utilizing a previously "non-functional" protein surface.

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