Article
Common patterns in type II restriction enzyme binding sites.
Institute of Molecular Biotechnology Beutenbergstrasse 11, D-07745 Jena, Germany.
Nucleic Acids Research (impact factor:
8.03).
02/2005;
33(8):2726-33.
DOI:10.1093/nar/gki575
pp.2726-33
Source: PubMed
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Article: Structure and function of type II restriction endonucleases.
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ABSTRACT: More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.Nucleic Acids Research 10/2001; 29(18):3705-27. · 8.03 Impact Factor -
Article: Crystallographic and bioinformatic studies on restriction endonucleases: inference of evolutionary relationships in the "midnight zone" of homology.
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ABSTRACT: Type II restriction endonucleases (ENases) cleave DNA with remarkable sequence specificity. Their discovery in 1970 and studies on molecular genetics and biochemistry carried out over the past four decades laid foundations for recombinant DNA techniques. Today, restriction enzymes are indispensable tools in molecular biology and molecular medicine and a paradigm for proteins that specifically interact with DNA as well as a challenging target for protein engineering. The sequence-structure-function relationships for these proteins are therefore of central interest in biotechnology. However, among numerous ENase sequences, only a few exhibit statistically significant similarity in pairwise comparisons, which was initially interpreted as evidence for the lack of common origin. Nevertheless, X-ray crystallographic studies of seemingly dissimilar type II ENases demonstrated that they share a common structural core and metal-binding/catalytic site, arguing for extreme divergence rather than independent evolution. A similar nuclease domain has been also identified in various enzymes implicated in DNA repair and recombination. Ironically, following the series of crystallographic studies suggesting homology of all type II ENases, bioinformatic studies provided evidence that some restriction enzymes are in fact diverged members of unrelated nuclease superfamilies: Nuc, HNH and GIY-YIG. Hence, the restriction enzymes as a whole, represent a group of functionally similar proteins, which evolved on multiple occasions and subsequently diverged into the "midnight zone" of homology, where common origins within particular groups can be inferred only from structure-guided comparisons. The structure-guided approaches used for this purpose include: identification of functionally important residues using superposition of atomic coordinates, alignment of sequence profiles enhanced by secondary structures, fold recognition, and homology modeling. This review covers recent results of comparative analyses of restriction enzymes from the four currently known superfamilies of nucleases with distinct folds, using crystallographic and bioinformatic methods, with the emphasis on theoretical predictions and their experimental validation by site-directed mutagenesis and biochemical analyses of the mutants.Current Protein and Peptide Science 11/2003; 4(5):327-37. · 2.89 Impact Factor -
Article: Chimeric restriction enzymes: what is next?
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ABSTRACT: Chimeric restriction enzymes are a novel class of engineered nucleases in which the non-specific DNA cleavage domain of Fokl (a type IIS restriction endonuclease) is fused to other DNA-binding motifs. The latter include the three common eukaryotic DNA-binding motifs, namely the helix-turn-helix motif, the zinc finger motif and the basic helix-loop-helix protein containing a leucine zipper motif. Such chimeric nucleases have been shown to make specific cuts in vitro very close to the expected recognition sequences. The most important chimeric nucleases are those based on zinc finger DNA-binding proteins because of their modular structure. Recently, one such chimeric nuclease, Zif-QQR-F(N) was shown to find and cleave its target in vivo. This was tested by microinjection of DNA substrates and the enzyme into frog oocytes (Carroll et al., 1999). The injected enzyme made site-specific double-strand breaks in the targets even after assembly of the DNA into chromatin. In addition, this cleavage activated the target molecules for efficient homologous recombination. Since the recognition specificity of zinc fingers can be manipulated experimentally, chimeric nucleases could be engineered so as to target a specific site within a genome. The availability of such engineered chimeric restriction enzymes should make it feasible to do genome engineering, also commonly referred to as gene therapy.Biological Chemistry 380(7-8):841-8. · 2.96 Impact Factor
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Keywords
acceptor clusters
DNA binding proteins
DNA recognition sites
enhanced GC content
features
general patterns
phage genomes
pyrimidines
recognition sites
Restriction enzymes
RR/YY dinucleotides
specific protein-DNA interactions
striking accumulation
studied examples
type II restriction endonucleases
type II restriction enzymes
