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LAGLIDADG Homing Endonuclease I-CreI

Christian Hinderer '10 and Nora Pencheva '09


I. Introduction

Homing endonucleases comprise a diverse family of proteins that are found in organisms from all branches of life: eubacteria, archaea, and eukaryotes. All homing endonucleases are characterized by their ability to laterally transfer their encoding sequence to a homologous allele lacking the sequence, a process termed homing. The homing endonucleases’ open reading frames (ORFs) are found within genetically mobile host introns. The intron elements carrying the homing ORFs can self-splice at the mRNA level, preventing any disruption of the host genetic information. Each homing endonuclease catalyzes the lateral transfer of its ORF by recognizing and cleaving a specific sequence (known as the homing site) embedded within a homologous allele that lacks the endonuclease-encoding intron. Following endonucleolytic cleavage of the homing site, the homing endonuclease encoding sequence can be laterally transferred and embedded within the DNA homing site via two different routes. Mobile group I introns utilize homologous recombination between alleles via a double-strand repair/gene conversion mechanism. Mobile group II introns use a reverse transcription mechanism to synthesize new DNA using the homing endonuclease mRNA sequence as a template (Belfort et al., 1995; Jurica et al., 1998; Chevalier and Stoddard, 2001).

 Homing endonucleases are organized in four general families that include the LAGLIDADG, His-Cys Box, HNH, and GIY-YIG family. The largest family of endonucleases (with more than 200 member proteins) is characterized by the presence of a conserved LAGLIDADG  residues motif (Chevalier and Stoddard, 2001). The protein I-CreI is a member of the LAGLIDADG family and it is among the best biochemically understood homing endonucleases. I-CreI is encoded by a group I intron found in the Chlamydomonas reinardtii chloroplast 23S rRNA gene (Jurica et al., 1998). I-CreI recognizes a target homing site that represents a 22-bp long pseudo-palindromic DNA sequence. Cleavage of the homing site by I-CreI generates two four-nucleotide long 3’ extended "sticky ends" . The cleavage reaction is catalyzed by three divalent cations bound at and between the enzyme active sites (Chevalier et al., 2001; Chevalier et al., 2004).

Given their ability of highly selective long-range DNA recognition and efficient cleavage, homing endonucleases have emerged as ideal models for the design of accurate enzymes for DNA cleavage and recombination. Site-specific mutations of residues in the DNA-binding regions of homing endonucleases, whose structures are known, have greatly expanded the range of potential homing targets available (Arnould et al., 2006). For instance, Redondo et al. (2008) recently reported two engineered structural derivatives of I-CreI that were successfully designed to recognize and cleave target DNA from the human xeroderma pigmentosum group C (XPC) gene. These findings confirm the utility of homing endonucleases, such as I-CreI, in providing novel insights into genome engineering and gene therapy.

II. General Structure of Endonuclease/DNA Complex

I-CreI is a homodimer composed of two symmetrical monomers. Each monomer contains five alpha helices lying on one face of a broad beta ribbon region comprised of four antiparallel beta strands. The association of the alpha and beta regions is mediated by contacts between four of the helices (a2, a3, a4, a5) and the internal face of the beta ribbon. The external face of the beta ribbon forms the DNA binding region. The fifth helix (a1), containing the first seven residues (13-19) of the LAGLIDADG motif (residues 13-21), lies perpendicular to the other helices at the interface of the two monomers. Residues 16 and 17 deviate from the consensus LAGLIDADG sequence such that in I-CreI Phe-16 and Val-17 replace the conserved Leu-16 and Ileu-17 in the consensus motif. Short range Van der Waals forces mediate the close interaction between the internal a1 helices of the two monomers. This interaction results in the juxtaposition of a conserved aspartate residue (Asp 20) from each monomer. The two Asp residues bind three divalent cations at the protein-DNA interface, forming the active site of the enzyme near the DNA minor groove. Conformational changes in the DNA are induced by I-CreI binding, which slightly bends the helix and compresses the minor groove adjacent to the enzyme’s active site (Jurica et al., 1998; Chevalier et al., 2001; Chevalier et al., 2003).  

III. DNA Binding

I-CreI interacts with the DNA homing site through a network of multiple direct contacts. The residues mediating DNA binding lie within the beta ribbon region of each I-CreI monomer. Each monomer utilizes eight residues of this region to recognize a nine-base pair DNA sequence. The beta ribbons curve along the DNA major groove allowing for a longer recognition sequence than it is generally possible with less flexible binding motifs such as a helix-turn-helix motif The distinctive curvature of the ribbon is dependent upon the loop connecting b1 and b2, which also contributes to DNA binding through three residues (Asn-30, Ser-32, and Tyr-33) that hydrogen bond with the DNA bases T, A, and G, respectively Sequence-specific DNA recognition by the beta ribbons is mediated by residues Arg-70, Gln-44, Arg-68, Gln-26, Lys-28, Gln-38, Tyr-33, and Ser-32, which project from b1, b2, and b4. Three of the most conserved nucleotides in the homing sequence are those that are recognized by Arg-68, Arg-70 and Gln-38; each protein residue makes double H-bond contacts with its respective DNA base. Nonspecific contacts to the DNA backbone are made by residues Lys-48, Arg-51, Lys-98, Ser-138, and Lys-116. Indirect contacts between the DNA minor groove, separating the homing sequence half sites, and enzyme residues Gln-47 and Asp-20 are made through water-mediated or metal-coordinated binding. (Jurica et al., 1998; Chevalier et al., 2001; Chevalier et al., 2003).

IV. Endonuclease Active Site

When bound by I-CreI, DNA is bent and the minor groove compressed, bringing the two scissile phosphate groups at the active site into close proximity to each other. The catalytic activity of I-CreI requires binding of three Mg2+ ions in the enzyme’s active site. In the uncleaved substrate state, the enzyme is complexed with inactivating Ca2+ ions. The most highly conserved residues in the active site, the C-terminal aspartate residues (Asp-20 and Asp-20') of the LAGLIDADG motif, directly stabilize the catalytic metal ions at the enzyme-DNA interface. Three other active site residues (Lys 98, Arg 51, and Gln-47) are also believed to be important for cleavage activity, though their role is not fully understood.

The three divalent cations bound in the enzyme’s active site form a line along the minor groove, positioning the two outer metal ions against the two scissile phosphate groups of each active site and leaving the third ion directly between the two active sites of the endonuclease. This arrangement allows for the central metal ion to participate in the catalytic activity of both of the outer ions. Each outer metal ion bound in the I-CreI active site is coordinated by an octahedral network of six ligands: the oxygen atom of the conserved Asp-20 residue, the carbonyl oxygen of Gly-19, two non-bridging oxygen atoms of a scissile phosphate and a non-scissile phosphate on the opposite DNA strand, and two water molecules. The central metal ion is also coordinated in an octahedral arrangement, with each monomer contributing three ligands: an oxygen atom from the Asp-20 residue, a 3’ bridging oxygen atom from a scissile phosphate, and a non-bridging oxygen from the scissile phosphate. (Jurica et al., 1998; Chevalier et al., 2001; Chevalier et al., 2003). 

V. DNA Cleavage

DNA cleavage occurs across the minor groove between the two scissile phosphate groups. The phosphodiester bond is hydrolyzed by an Sn2 mechanism. The HO- nucleophile is stabilized and positioned for attack by the outer Mg2+ ion adjacent to a scissile phosphate. The 3’ oxyanion leaving group is then stabilized by the internal Mg2+ ion following DNA strand cleavage. The three metal ions remain bound in the cleaved complex, with the outer ions now coordinated with a hydroxyl group attached to the 5’ phosphate rather than a water molecule. The resulting double-strand break, consisting of two four-nucleotide long 3’ cohesive ends, can serve as a trigger of homologous recombination that will ultimately transfer the ORF encoding I-CreI to the cleaved homing DNA site. (Chevalier et al., 2001; Chevalier et al., 2003). 

VI. References

Arnould, S., Chames, P., Perez, C., Lacroix, E., Dulcert, A., Epinat, J.C., Stricher, F., Petit, A.S., Patin, A., Guillier, S., Rolland, S., Prieto, J., Blanco, F.J., Bravo, J., Montoya, G., Serrano, L., Duchateau, P., Paques, F. 2006.  Engineering of large numbers of highly specific homing endonucleases that induce recombination on novel DNA targets. J. Mol. Biol.: 355, 443-458.

Belfort, M. and Perlman, P.S. 1995. Mechanisms of intron mobility. J. Biol. Chem.: 270, 30237-30240.

Chevalier, B., Monique, T., Lemieux, C., Monnat, R.J., Stoddard, B.L.  2003. Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI. .J.  Mol.  Biol.: 329, 253-269.

Chevalier, B.S., Monnat, R.J., Stoddard, B.L. 2001. The homing endonuclease I-CreI uses three metals, one of which is shared between the two active sites. Nature Struct. Biol.:8, 312-315.

Chevalier, B.S., Stoddard, B.L. 2001. Homing endonucleases: structural and functional insight into the catalysis of intron/intein mobility. Nucl. Ac. Res.: 18, 3757-3774.

Chevalier, B.S., Sussman, D., Otis, C., Nol, A., Turmel, M., Lemieux, M., Stephens, K., Monnat, R., Stoddard, B.L. 2004. Metald-dependent DNA cleavage mechanism of the I-CreI LAGLIDADG homing endonuclease.  Biochemistry: 43, 14015-14026.

Jurica, M.S., Monnat, R., Stoddard, B.L. 1998. DNA Recognition and Cleavage by the LAGLIDADG Homing Endonuclease I-CreI. Molecular Cell: 2, 469-476.

Redondo, P., Prieto, J., Munoz, I.G., Alibes, A., Stricher, F., Serrano, L., Cabaniols, J.P., Daboussi, F., Arnold, S., Perez, C., Duchateau, P., Paques, F., Blanco, F.J., Montoya, G. 2008.  Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases. Nature: 456: 107-111.

Stoddard, B.L. 2006. Homing endonuclease structure and function. Quart. Rev. Biophys.: 38, 49-95.

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