A long-term goal in the field of restrictionmodification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create "artificial" nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.Since their discovery nearly 25 years ago (1), type II restriction enzymes have played a crucial role in the development of the recombinant DNA technology and the field of molecular biology. The type II restriction endonucleases and modification methylases are relatively simple bacterial enzymes that recognize specific sequences in duplex DNA. While the former cleave DNA, the latter methylate adenine or cytosine residues within the recognition site so as to protect the host genome against cleavage. So far, over 2500 restriction-modification (R-M) enzymes have been identified, and these are found in widely diverse organisms (2). These enzymes fall into numerous "isoschizomer" (identically cleaving) groups with about 200 sequence specificities. Discovery of new enzymes involves tedious and time-consuming effort that requires extensive screening of bacteria and other microorganisms (3). Even when one finds a new enzyme, more often than not, it falls into the already discovered isoschizomer groups. Furthermore, most naturally occurring restriction enzymes recognize sequences that are 4-6 bp long. Although these enzymes are very useful in manipulating recombinant DNA, they are not suitable for producing large DNA segments. For example, restriction enzymes that recognize DNA sequences 6 bp long result in cuts as often as every 4096 bases. In many instances, it is preferable to have fewer but longer DNA strands, especially during genome mapping. Rare cutters that recognize 8-bp-long sequences cut human DNA (which contains about 3 billion bp) every 65,536 bases on average. So far, only a few restriction endonucleases with recognition sequences longer than 6 bp (rare cutters) have been identified (New England Biolabs catalog). R-M systems appear to have a single biological function-namely, to protect cells from infection by foreign DNA that would otherwise destroy them. The phage genomes are usually small. It stands to reason, then, that bacteria select for R-M systems with small recognition sites (4-6 bp) because these sites occur more frequently in the phages. Therefore, aThe publication costs of this article were defrayed in part by page charge ...

Chimeric nucleases that are hybrids between a nonspecific DNA cleavage domain and a zinc finger DNA recognition domain were tested for their ability to find and cleave their target sites in living cells. Both engineered DNA substrates and the nucleases were injected into Xenopus laevis oocyte nuclei, in which DNA cleavage and subsequent homologous recombination were observed. Specific cleavage required two inverted copies of the zinc finger recognition site in close proximity, reflecting the need for dimerization of the cleavage domain. Cleaved DNA molecules were activated for homologous recombination; in optimum conditions, essentially 100% of the substrate recombined, even though the DNA was assembled into chromatin. The original nuclease has an 18-amino-acid linker between the zinc finger and cleavage domains, and this enzyme cleaved in oocytes at paired sites separated by spacers in the range of 6 to 18 bp, with a rather sharp optimum at 8 bp. By shortening the linker, we found that the range of effective site separations could be narrowed significantly. With no intentional linker between the binding and cleavage domains, only binding sites exactly 6 bp apart supported efficient cleavage in oocytes. We also showed that two chimeric enzymes with different binding specificities could collaborate to stimulate recombination when their individual sites were appropriately placed. Because the recognition specificity of zinc fingers can be altered experimentally, this approach holds great promise for inducing targeted recombination in a variety of organisms.Procedures and reagents that allow the directed alteration of genes in situ constitute a powerful toolbox for experimental genetics and potentially for agricultural and therapeutic applications. In many organisms, however, and particularly in higher eukaryotes, the efficiency of recombination between an introduced DNA and the homologous chromosomal target is discouragingly low. For example, such events typically occur in mammalian cells at a frequency of only about 1 for each 10 6 cells treated (3, 31). We are interested in developing procedures that would substantially improve the frequency of gene targeting.A major impediment to efficient gene replacement is the status of the chromosomal target. Increasing the number of target sequences has little or no effect on targeting efficiency (54, 60). In contrast, making an intentional double-strand break (DSB) in the target DNA increases the yield of specific homologous recombination events up to 1,000-fold or more (10,11,14,44,46). It is believed that exonucleases act at broken ends to generate single-stranded tails that are recombinagenic in any of several pathways. In particular, the singlestrand annealing mechanism (33), by which homologous recombination involving exogenous DNA usually occurs in higher eukaryotes (53), cannot proceed unless both the donor and target have ends (5, 48).Whatever the mechanism of recombination, it is clear that the frequency of targeted recombination can be substantially improved by i...

Left-handed Z-DNA is a higher-energy form of the double helix, stabilized by negative supercoiling generated by transcription or unwrapping nucleosomes. Regions near the transcription start site frequently contain sequence motifs favourable for forming Z-DNA, and formation of Z-DNA near the promoter region stimulates transcription. Z-DNA is also stabilized by specific protein binding; several proteins have been identified with low nanomolar binding constants. Z-DNA occurs in a dynamic state, forming as a result of physiological processes then relaxing to the right-handed B-DNA. Each time a DNA segment turns into Z-DNA, two B-Z junctions form. These have been examined extensively, but their structure was unknown. Here we describe the structure of a B-Z junction as revealed by X-ray crystallography at 2.6 A resolution. A 15-base-pair segment of DNA is stabilized at one end in the Z conformation by Z-DNA binding proteins, while the other end remains B-DNA. Continuous stacking of bases between B-DNA and Z-DNA segments is found, with the breaking of one base pair at the junction and extrusion of the bases on each side (Fig. 1). These extruded bases may be sites for DNA modification.

The N-terminal domain of the E3L protein of vaccinia virus has sequence similarity to a family of Z-DNA binding proteins of defined three-dimensional structure and it is necessary for pathogenicity in mice. When other Z-DNA-binding domains are substituted for the similar E3L domain, the virus retains its lethality after intracranial inoculation. Mutations decreasing Z-DNA binding in the chimera correlate with decreases in viral pathogenicity, as do analogous mutations in wild-type E3L. A chimeric virus incorporating a related protein that does not bind Z-DNA is not pathogenic, but a mutation that creates Z-DNA binding makes a lethal virus. The ability to bind the Z conformation is thus essential to E3L activity. This finding may allow the design of a class of antiviral agents, including agents against variola (smallpox), which has an almost identical E3L.

Editing of RNA changes the read-out of information from DNA by altering the nucleotide sequence of a transcript. One type of RNA editing found in all metazoans uses double-stranded RNA (dsRNA) as a substrate and results in the deamination of adenosine to give inosine, which is translated as guanosine. Editing thus allows variant proteins to be produced from a single pre-mRNA. A mechanism by which dsRNA substrates form is through pairing of intronic and exonic sequences before the removal of noncoding sequences by splicing. Here we report that the RNA editing enzyme, human dsRNA adenosine deaminase (DRADA1, or ADAR1) contains a domain (Z␣) that binds specifically to the left-handed Z-DNA conformation with high affinity (K D ‫؍‬ 4 nM). As formation of Z-DNA in vivo occurs 5 to, or behind, a moving RNA polymerase during transcription, recognition of Z-DNA by DRADA1 provides a plausible mechanism by which DRADA1 can be targeted to a nascent RNA so that editing occurs before splicing. Analysis of sequences related to Z␣ has allowed identification of motifs common to this class of nucleic acid binding domain.A well characterized editing mechanism affecting doublestranded RNA (dsRNA) involves the deamination of adenosine to produce inosine, which is translated as guanosine (1). This activity has been reported to be widespread throughout metazoa. The first example showing the physiological relevance of dsRNA editing in mammals was the replacement of a glutamine (CAG) by arginine (CGG) in the ion channel of a glutamate-responsive neuroreceptor (GluR). This change decreased the Ca 2ϩ permeability of the receptor (2-4). Subsequently, other examples of GluR RNA editing also have been identified (5, 6), as well as editing of the serotonin-2C receptor (7) and the 4f-rnp gene from Drosophila (8). So far, two types of enzymes have been reported that are capable of performing dsRNA editing in vitro, DRADA1 (the prototype of the ADAR1 family that includes dsRAD1 and dsRAD2) and RED1 (the prototype of the ADAR2 family that includes DRADA2) (9-15). A third protein, RED2, which has strong sequence homology to RED1, has as yet no known in vitro or in vivo substrate (17). RED1 was cloned using low-stringency hybridization with probes prepared from DRADA1. Both DRADA1 and RED-1 are present in all tissues tested, suggesting that dsRNA editing is a widespread process. However, these enzymes show differences in editing specificity when transiently coexpressed with RNA substrates in vivo (13,14). DRADA1 and RED1 are similar to each other in their catalytic and dsRNA binding motifs (9, 10, 13, 18), but differ in that the N terminus of DRADA1 contains domains absent from RED1. The possibility therefore exists that this difference in structure determines how RED1 and DRADA1 are used within cells. METHODSIdentification of the Z␣ Domain. The Z-DNA binding domain (Z␣) initially was mapped to the N terminus of DRADA1 by testing baculovirus-expressed protein and showing that band shift activity required the presence of residues 1-296. This...