Brett Chevalier

Design, Activity, and Structure of a Highly Specific Artificial Endonuclease

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.

Flexible DNA Target Site Recognition by Divergent Homing Endonuclease Isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.

The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites.

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.

The LAGLIDADG Homing Endonuclease Family

The LAGLIDADG protein family includes the first identified and biochemically characterized intron-encoded proteins (Dujon 1980; Lazowska et al. 1980; Jacquier and Dujon 1985), as described in this volume by Dujon. It has been variously termed the ‘DOD’, ‘dodecapeptide’, ‘dodecamer’, and ‘decapeptide’ endonuclease family, based on the conservation of a ten-residue sequence motif (Dujon 1989; Dujon et al. 1989; Belfort et al. 1995; Belfort and Roberts 1997; Dalgaard et al. 1997; Chevalier and Stoddard 2001). The LAGLIDADG endonucleases are the most diverse of the homing endonuclease families. Their host range includes the genomes of plant and algal chloroplasts, fungal and protozoan mitochondria, bacteria and Archaea (Dalgaard et al. 1997). One reason for the wide phylogenetic distribution of LAGLIDADG genes appears to be their remarkable ability to invade unrelated types of intervening sequences, including group I introns, archaeal introns and inteins (Belfort and Roberts 1997; Chevalier and Stoddard 2001). Descendents of LAGLIDADG homing endonucleases also include the yeast HO mating type switch endonuclease (Jin et al. 1997), which is encoded by an independent reading frame rather than within an intron, but does carry remnants of an inactive intein domain (Haber and Wolfe, this Vol.), and maturases that assist in RNA splicing (Delahodde et al. 1989; Lazowska et al. 1989; Schafer et al. 1994; Geese and Waring 2001; Caprara and Waring, this Vol.).