Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor

Bolduc, Jill M.; Spiegel, P. Clint; Chatterjee, Piyali; Brady, Kristina L.; Downing, Maureen E.; Caprara, Mark G.; Waring, Richard B.; Stoddard, Barry

doi:10.1101/gad.1109003

Cited by 88 publications

(93 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…S2A). Both proteins make contacts with approximately one-half of the nucleotide bases and backbone phosphate groups of their target sequences, via a mixture of direct and water-mediated contacts, as observed in previous LHE crystal structures (16)(17)(18) (Fig. 2B).…”

Section: Global Analyses Of Single-chain Laglidadg Enzymes Reveals DIsupporting

confidence: 66%

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Takeuchi

Lambert²,

Mak

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

110

View full text Add to dashboard Cite

Homing endonucleases mobilize their own genes by generating double-strand breaks at individual target sites within potential host DNA. Because of their high specificity, these proteins are used for “genome editing” in higher eukaryotes. However, alteration of homing endonuclease specificity is quite challenging. Here we describe the identification and phylogenetic analysis of over 200 naturally occurring LAGLIDADG homing endonucleases (LHEs). Biochemical and structural characterization of endonucleases from one clade within the phylogenetic tree demonstrates strong conservation of protein structure contrasted against highly diverged DNA target sites and indicates that a significant fraction of these proteins are sufficiently stable and active to serve as engineering scaffolds. This information was exploited to create a targeting enzyme to disrupt the endogenous monoamine oxidase B gene in human cells. The ubiquitous presence and diversity of LHEs described in this study may facilitate the creation of many tailored nucleases for genome editing.

show abstract

Section: Global Analyses Of Single-chain Laglidadg Enzymes Reveals DIsupporting

confidence: 66%

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Takeuchi

Lambert²,

Mak

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

110

View full text Add to dashboard Cite

show abstract

“…This analysis revealed a series of conserved basic residues distributed across the protein, at positions removed from the DNA binding site ( Fig. 1A; Bolduc et al 2003). A single glutamate substitution in one of the conserved residues (arginine 239 [R239E]) four residues from the C terminus resulted in a 10-fold decrease in A. nidulans COB pre-RNA splicing and ribonucleoprotein (RNP) dissociation rates.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, half of known maturases also retain homing activity (Dalgaard et al 1997;Lambowitz et al 1999). Finally, comparison of the threedimensional structures between a dual function maturase/ endonuclease from Aspergillus nidulans (I-AniI) with a number of other homing endonucleases shows that both types of proteins share a conserved overall topology (Bolduc et al 2003). While the mechanisms by which homing endonucleases recognize and cleave their target sequences are well established, how such proteins bind with high specificity to their intron RNA substrates and promote RNA splicing is only beginning to be uncovered (Ho and Waring 1999;Solem et al 2002).…”

Section: Introductionmentioning

confidence: 99%

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Downing

Brady²,

Caprara³

2005

RNA

Self Cite

View full text Add to dashboard Cite

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N-and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.

show abstract

“…The Ca 2ϩ atom is hexa-coordinated with phosphates from both DNA strands (2A strandA and Ϫ3C strandB ), 3 water molecules and D21 from the first LAGLI-DADG motif. The additional anomalous difference study in the presence of Mn 2ϩ demonstrated binding of 2 metal ions with equivalent occupancy, similarly to the Mg 2ϩ positions reported for I-AniI (20) (Fig. 2B and Fig.…”

Section: Resultsmentioning

confidence: 52%

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography M eganucleases are sequence-specific enzymes that recognize large (12-45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron-or intein-containing gene, resulting in the insertion of the intron or intein in that particular site (1). Meganucleases are being used to stimulate homologous gene targeting in the vicinity of their target sequences with the aim of improving current genome engineering approaches, alleviating the risks due to the randomly inserted transgenes (2, 3).The use of meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases. In nature, meganucleases are essentially represented by homing endonucleases, a family of enzymes encoded by mobile genetic elements whose function is to initiate DSB induced recombination events in a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely low. Thus, making artificial meganucleases with custom-made substrate specificity is an intense area of research.Sequence homology has been used to classify homing endonucleases into 5 families, the largest one having the conserved LAGLI-DADG sequence motif. Homing endonucleases containing one such motif function as homodimers. In contrast, homing endonucleases containing 2 motifs are single chain proteins (5, 6). Structural information for several members of the LAGLIDADG endonuclease family indicate that these proteins adopt a similar active conformation as homodimers or as monomers with 2 separate domains (7-9). The LAGLIDADG motifs form structurally conserved ␣-helices tightly packed at the center of the interdomain or intermonomer interface. The last acidic residue of this LAGLI-DADG motif part...

show abstract

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor

Cited by 88 publications

References 55 publications

Tapping natural reservoirs of homing endonucleases for targeted gene modification

Tapping natural reservoirs of homing endonucleases for targeted gene modification

A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Contact Info

Product

Resources

About