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

Chevalier, B.; Kortemme, Tanja; Chadsey, Meggen S.; Baker, David; Monnat, Raymond J.; Stoddard, Barry

doi:10.1016/s1097-2765(02)00690-1

Cited by 217 publications

(166 citation statements)

References 44 publications

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“…The DNA cleavage domain of the type IIS REase FokI was fused with DNA binding proteins such as the Drosophila Ubx homeodomain (27), the yeast Gal4 protein (28) and zinc-finger proteins (29) to engineer chimeric endonucleases. Furthermore, it was demonstrated that domains from unrelated homing endonucleases or mutated subdomains of an individual homing enzyme can be fused to create active chimeric enzymes of altered specificity (30)(31)(32)(33). However, the hybrids mentioned above are extremely rarely cutting enzymes, which have specific applications including gene therapy (34,35), but their potential to be used as analytical tools like conventional type II REases is limited.…”

Section: Discussionmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

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

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Type II restriction endonucleases (REases) cleave double-stranded DNA at specific sites within or close to their recognition sequences. Shortly after their discovery in 1970, REases have become one of the primary tools in molecular biology. However, the list of available specificities of type II REases is relatively short despite the extensive search for them in natural sources and multiple attempts to artificially change their specificity. In this study, we examined the possibility of generating cleavage specificities of REases by swapping putative target recognition domains (TRDs) between the type IIB enzymes AloI, PpiI, and TstI. Our results demonstrate that individual TRDs recognize distinct parts of the bipartite DNA targets of these enzymes and are interchangeable. Based on these properties, we engineered a functional type IIB REase having previously undescribed DNA specificity. Our study suggests that the TRD-swapping approach may be used as a general technique for the generation of type II enzymes with predetermined specificities.hybrid ͉ AloI ͉ PpiI ͉ TstI R estriction endonucleases (REases) are parts of restrictionmodification (R-M) systems, whose primary biological function is the protection of bacterial cells from incoming foreign DNA molecules (1). There are three main groups of restriction enzymes (types I, II, and III), which differ in enzyme composition, cofactor requirements, and mode of action (2). The beststudied are type II REases, which in general recognize specific DNA targets of 4-8 bp and cleave DNA at or close to these sequences (1, 2). The exquisite accuracy of type II enzymes (3, 4) has made them indispensable tools for DNA manipulations. Although almost 3,700 type II REases with 262 different specificities have been characterized to date (5), there still is a demand for enzymes recognizing new DNA targets.During the past two decades, numerous efforts have been undertaken to engineer type II REases with altered specificities. Both rational protein design and random mutagenesis, followed by various selection procedures, have been tried (6), and several mutant enzymes with some preference for cleavage of altered DNA targets were isolated (7-10). However, projects concerned with orthodox type II REases so far have been largely unsuccessful mainly for two reasons: (i) difficulty of dealing with the observed tight coupling between DNA recognition and cleavage and (ii) absence of an efficient system for selecting enzymes with changed specificities. In this regard, unorthodox type II enzymes, such as the type IIG REase Eco57I (11), have shown more promise. Type IIG enzymes combine the catalytic centers of endonuclease and methyltransferase in one polypeptide chain, and the ability of Eco57I to methylate recognized DNA targets has been applied to isolate mutants having previously undescribed specificity (12).The discovery of AloI-like REases (13-15), classified as type IIB enzymes, opened up new opportunities for the engineering of type II REases with altered specificities. AloI-like REases ar...

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Section: Discussionmentioning

confidence: 99%

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Jurenaite-Urbanaviciene

Serksnaite²,

Kriukienė

et al. 2007

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

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“…Thus a sequence-specific nuclease or targeted DNA damage would be the basis of a strategy for manipulation of the genome. Although considerable effort has been given to the development of chimeric nucleases for this purpose (17,18), the alternate approach based on targeted DNA damage has received much less attention (9,19).…”

mentioning

confidence: 99%

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

Majumdar

Muniandy

Liu

et al. 2008

Journal of Biological Chemistry

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Information from exogenous donor DNA can be introduced into the genome via homology-directed repair (HDR) pathways. These pathways are stimulated by double strand breaks and by DNA damage such as interstrand cross-links. We have employed triple helix-forming oligonucleotides linked to psoralen (pso-TFO) to introduce a DNA interstrand cross-link at a specific site in the genome of living mammalian cells. Co-introduction of duplex DNA with target region homology resulted in precise knock in of the donor at frequencies 2-3 orders of magnitude greater than with donor alone. Knock-in was eliminated in cells deficient in ERCC1-XPF, which is involved in recombinational pathways as well as cross-link repair. Separately, single strand oligonucleotide donors (SSO) were co-introduced with the pso-TFO. These were 10-fold more active than the duplex knock-in donor. SSO efficacy was further elevated in cells deficient in ERCC1-XPF, in contrast to the duplex donor. Resected single strand ends have been implicated as critical intermediates in sequence modulation by SSO, as well as duplex donor knock in. We asked whether there would be a competition between the donor species for these ends if both were present with the pso-TFO. The frequency of duplex donor knock in was unaffected by a 100-fold molar excess of the SSO. The same result was obtained when the homing endonuclease I-SceI was used to initiate HDR at the target site. We conclude that the entry of double strand breaks into distinct HDR pathways is controlled by factors other than the nucleic acid partners in those pathways. Double strand breaks (DSBs)2 are among the most dangerous forms of DNA damage and may result in deletion, rearrangement of chromosomal sequences, or, if unrepaired, chromosome loss and possibly cell death (1). There are multiple pathways for DSB repair that are distinguished by the identity of the proteins and enzymes involved and their potential for mutagenesis of the break site (2-8). Non-homologous end joining, the major pathway in mammalian cells, is homology-independent and often results in small deletions at the site of the break. There are two homology-directed repair (HDR) pathways in which resected single-stranded ends interact with homologous sequences. In single strand annealing (SSA), the single strand end anneals with a complementary single strand. SSA between two direct repeated sequences results in the retention of one copy of the repeat and deletion of the other copy and the intervening sequence. Homologous recombination repair involves invasion of a DNA duplex by a single strand end and can be error-free or mutagenic depending on the sequence of the invaded duplex. Because the sequences with which they interact need not be absolutely identical to the single strand end, the homology-directed pathways provide an opportunity to manipulate the sequence of the genome.Gene conversion and recombination with exogenous double and single strand donor DNAs occur at impractically low frequencies in mammalian cells (9). However, DSBs in the genom...

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“…S4). In contrast, 3 Mg 2ϩ were reported for I-CreI (17) and H-DreI active sites (15), even though in the last case the DNA remained intact. Two of the metal ions were unshared, whereas the central one was shared between the 2 catalytic residues (Fig.…”

Section: Resultsmentioning

confidence: 92%

“…The structure of the I-DmoI/DNA complex also depicts differences when compared with H-DreI (Fig. S2) (15), a chimerical enzyme that contains the domain A of I-DmoI fused to an I-CreI monomer (originally named E-DreI). A comparison between the wild type structure and the I-DmoI domain A of the chimera reveals subtle changes in the DNA conformation (see below).…”

Section: Resultsmentioning

confidence: 99%

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.

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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...

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

Cited by 217 publications

References 44 publications

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Generation of DNA cleavage specificities of type II restriction endonucleases by reassortment of target recognition domains

Targeted Gene Knock In and Sequence Modulation Mediated by a Psoralen-linked Triplex-forming Oligonucleotide*

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

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