DNA determinants important in sequence recognition by Eco RI endonuclease

Alkylation interference and protection methods (Siebenlist, U., and Gilbert, W., (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 122-126) have been utilized to deduce potential DNA contacts involved in specific complex formation between Eco RI endonuclease and its recognition sequence. The endonuclease protected the N7 position (major groove) of the dG and the N3 position (minor groove) of both dA residues within the Eco RI sequence against alkylation by dimethylsulfate, d(GpApApTpTpC), suggesting the presence of poly-peptide in both grooves in the vicinity of affected nitrogens. Results of methylation interference analysis suggest that the N7 of the Eco RI site dG and the N3 of the central dA, d(GpApApTpTpC), are utilized as contacts by the enzyme. The failure to observe interference upon methylation of the 5'-penultimate dA within the sequence implies that the endonuclease does not bond to the N3 of this residue, despite the fact that it is protected against alkylation by the protein. Ethylation interference patterns suggest four major phosphate contacts between endonuclease and each DNA strand. Two of these phosphates are 5'-external to the Eco RI sequence, d(pNpGpApApTpTpC), suggesting involvement of outside phosphates in electrostatic interactions. Moreover, alkylation protection and interference effects on the two DNA strands display perfect 2-fold symmetry. Thus, the endonuclease interacts with a minimum of 10 nucleotide pairs to yield a DNA-protein complex characterized by elements of symmetry. In contrast, specific alkylation effects were not observed in comparable experiments with the endonuclease and a DNA which had been previously methylated by the Eco RI modification enzyme.     

Prediction of secondary structure for Eco RI endonuclease

The circular dichroism of Eco RI restriction endonuclease was measured to 178 nm and analyzed for secondary structure. The results (33% alpha-helix, 25% beta-sheet, 17% turns, and 25% other structures) compare well with our joint prediction from sequence data.     

Kinetic studies on the cleavage of adenovirus DNA by restriction endonuclease Eco RI.

The kinetics of cleavage of DNA from Adenovirus Type 1 (Ad1), Type 5 (Ad5) and Type 6 (Ad6) by restriction endonuclease EcoRI was investigated by quantitative evaluation of the fluorescence from ethidium stained DNA fragments separated on agarose gels. The apparent rate constants of cleavage at different cleavage sites have been determined and large differences in the cleavage rates of the individual sites within one type of DNA were found. From the kinetics of cleavage information on the sequence of the DNA fragments can be obtained. The order of the fragment A, B, C, D of Ad6 DNA obtained after complete cleavage by restriction endonuclease Eco RI was found to be A-D-C-B; the order of the corresponding fragments A, B, C of Ad1 and Ad5 DNA was found to be A-C-B.     

Substrate specificity of restriction endonuclease Eco781

The recognition sequence and cleavage point of restriction endonuclease Eco781 have been determined as 5'-GGCGCC-. There are several known enzymes recognizing the same sequence, although the prototype NarI and isoschizomers NdaI and NunII cleave the substrate to produce 5'-protruding ends, whereas cleavage with isoschizomer BbeI results in 3'-protruding ends. Therefore, restrictase Eco78I, generating flush ends, may be regarded as an enzyme with new specificity among the restriction endonucleases recognizing the 5'-GGCGCC-sequence.     

Mechanisms of coupling between DNA recognition specificity and catalysis in EcoRI endonuclease

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.     

Use of affinity chromatography for the purification of specific endonuclease Eco RI and Bg1 II

The highly active preparations of specific endonucleases Eco RI and Bgl II were purified by affinity chromatography from E. coli and Bacillus globiggii cells, respectively. The isolation and purification procedures included cell disruption by ultrasonication, ultracentrifugation and chromatography. Blue dextrane-Sepharose, folate-Sepharose and phenyl-Sepharose were used as affinity adsorbents. The optimal conditions for the adsorption and elution of the endonucleases excluding intermediate steps of dialysis and concentration were selected. A high degree of purification was achieved by a consecutive use of adsorbents with different ligands. The purified enzyme does not contain non-specific nucleases or phosphatases, is sufficiently concentrated and can be used for specific hydrolysis of DNA.     

Profile of the DNA recognition site of the archaeal homing endonuclease I-DmoI.

I- Dmo I is a homing enzyme of the LAGLI-DADG type that recognizes up to 20 bp of DNA and is encoded by an archaeal intron of the hyperthermophilic archaeon Desulfurococcus mobilis . A combined mutational and DNA footprinting approach was employed to investigate the specificity of the I- Dmo I-substrate interaction. The results indicate that the enzyme binds primarily to short base paired regions that border the sites of DNA cleavage and intron insertion. The minimal substrate spans no more than 15 bp and while sequence degeneracy is tolerated in the DNA binding regions, the sequence and size of the cleavage region is highly conserved. The enzyme has a slow turnover rate and cuts the coding strand with a slight preference over the non-coding strand. Complex formation produces some distortion of the DNA double helix within the cleavage region. The data are compatible with the two DNA-binding domains of I- Dmo I bridging the minor groove, where cleavage occurs, and interacting within the major groove on either side, thereby stabilizing a distorted DNA double helix. This may provide a general mode of DNA interaction at least for the LAGLIDADG-type homing enzymes.     

Effect of temperature and 4,6'-diamidine-2-phenylindole on restriction of supercoiled Col E1 DNA by Eco RI endonuclease.

Supercoiled Col E1 DNA is split by Eco RI endonuclease at 37 degrees C without intermediate formation of open circular DNA. Accumulation of this restriction product is observed at low temperature. The fluorescent dye, 4,6'-diamidine-2-phenylindole (DAPI) inhibits restriction by Eco RI endonuclease. This effect is due to the DAPI:DNA rather than to the DAPI:Eco RI interactions.     

Methylation of Eco RI (GAm'ATTC) sequences of bacterial DNA

The methylation of Eco RI (GAm6 ATTC) sequences of DNA of bacteria related to the main branches of their phylogenetic dendrogramme, was studied. It was found that methylation of Eco RI sites is observed in bacteria Caulobacter and in Thermus aquaticus. This finding can be substantiated by the resistance of these DNAs to Eco RI restrictase as well as by the fact that Bam HI fragments of these DNAs cloned within the composition of the vector plasmid pUC8 in E. coli cells contain GAATTC sites.     

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.     

Evolution of I-SceI homing endonucleases with increased DNA recognition site specificity

Elucidating how homing endonucleases undergo changes in recognition site specificity will facilitate efforts to engineer proteins for gene therapy applications. I-SceI is a monomeric homing endonuclease that recognizes and cleaves within an 18-bp target. It tolerates limited degeneracy in its target sequence, including substitution of a C:G₊₄ base pair for the wild-type A:T₊₄ base pair. Libraries encoding randomized amino acids at I-SceI residue positions that contact or are proximal to A:T₊₄ were used in conjunction with a bacterial one-hybrid system to select I-SceI derivatives that bind to recognition sites containing either the A:T₊₄ or the C:G₊₄ base pairs. As expected, isolates encoding wild-type residues at the randomized positions were selected using either target sequence. All I-SceI proteins isolated using the C:G₊₄ recognition site included small side-chain substitutions at G100 and either contained (K86R/G100T, K86R/G100S and K86R/G100C) or lacked (G100A, G100T) a K86R substitution. Interestingly, the binding affinities of the selected variants for the wild-type A:T₊₄ target are 4- to 11-fold lower than that of wild-type I-SceI, whereas those for the C:G₊₄ target are similar. The increased specificity of the mutant proteins is also evident in binding experiments in vivo. These differences in binding affinities account for the observed ∼36-fold difference in target preference between the K86R/G100T and wild-type proteins in DNA cleavage assays. An X-ray crystal structure of the K86R/G100T mutant protein bound to a DNA duplex containing the C:G₊₄ substitution suggests how sequence specificity of a homing enzyme can increase. This biochemical and structural analysis defines one pathway by which site specificity is augmented for a homing endonuclease.     

An asymmetric complex of restriction endonuclease MspI on its palindromic DNA recognition site

Most well-known restriction endonucleases recognize palindromic DNA sequences and are classified as Type IIP. Due to the recognition and cleavage symmetry, Type IIP enzymes are usually found to act as homodimers in forming 2-fold symmetric enzyme-DNA complexes. Here we report an asymmetric complex of the Type IIP restriction enzyme MspI in complex with its cognate recognition sequence. Unlike any other Type IIP enzyme reported to date, an MspI monomer and not a dimer binds to a palindromic DNA sequence. The enzyme makes specific contacts with all 4 base pairs in the recognition sequence, by six direct and five water-mediated hydrogen bonds and numerous van der Waal contacts. This MspI-DNA structure represents the first example of asymmetric recognition of a palindromic DNA sequence by two different structural motifs in one polypeptide. A few possible pathways are discussed for MspI to cut both strands of DNA, either as a monomer or dimer.     

Increased chromosomal radiosensitivity of a Chinese hamster ovary cell line that inducibly expresses the Eco RI restriction endonuclease

We have transfected a Chinese hamster ovary cell line (CHO 6) with a plasmid that inducibly expresses the Eco RI restriction endonuclease gene in the presence of cadmium sulfate (CdSO4). Expression of Eco RI results in DNA double-strand breaks, which can lead to chromosome aberrations. The new line, designated CHO 10, also has a low level of constitutive expression of Eco RI in the absence of CdSO4 without any cytogenetic effect. This suggested that these cells may be efficient at repairing low levels of DNA double-strand breaks. To test this, both cell lines were exposed to ionizing radiation, and aberration yields were analyzed with or without induction of Eco RI. CHO 10 cells showed increased radiosensitivity after G1 irradiation, but after G2 exposure, only doses greater than or equal to 0.4 Gy caused more damage in CHO 10 cells. We conclude that CHO 10 cells can tolerate constitutive expression of Eco RI, but that when the cells are subjected to additional stress, in this case ionizing radiation, they become very sensitive to DNA double-strand breaks.     

DNA structure specificity of Rap endonuclease.

The Rap protein of phage lambda is an endonuclease that nicks branched DNA structures. It has been proposed that Rap can nick D-loops formed during phage recombination to generate splice products without the need for the formation of a 4-strand (Holliday) junction. The structure specificity of Rap was investigated using a variety of branched DNA molecules made by annealing partially complementary oligo-nucleotides. On Holliday junctions, Rap endonuclease shows a requirement for magnesium or manganese ions, with Mn(2+)supporting 5-fold more cleavage than Mg(2+). The location of endonuclease incisions was determined on 3'-tailed D-loop, bubble, flayed duplex, 5'-flap and Y junction DNA substrates. In all cases, Rap preferentially cleaves at the branch point of these molecules. With a flayed duplex, incisions are made in the duplex adjacent to the single-strand arms. Comparison of binding and cleavage specificities revealed that Rap is highly structure-specific and exhibits a clear preference for 4- and 3-stranded DNA over Y and flayed duplex DNA. Almost no binding or cleavage was detected with duplex, partial duplex and single-stranded DNA. Thus Rap endonuclease shows a bias for structures that resemble D-loop and Holliday junction recombination intermediates.     

Processive cleavage of concatemer DNA duplexes by Eco RII restriction endonuclease

Eco RII restriction endonuclease cleaves synthetic DNA-duplexes in which the recognition sites of this enzyme (5..CC _T ^A GG...) are repeated every 9 base pairs with the alternating orientation of the central AT pair. It operates in a processive mode, i.e. the bound enzyme molecule slides along the substrate toward neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained point to the capability of Eco RII endonuclease to recognize and cleave the substrate under both possible orientations of the central AT-pair of the recognition site with respect to the bound enzyme molecule. These data also show the close similarity of DNA structures in a complex with theenzyme and without.     

DNA distortion and specificity in a sequence-specific endonuclease

Five new structures of the Q138F HincII enzyme bound to a total of three different DNA sequences and three different metal ions (Ca²⁺, Mg²⁺, and Mn²⁺) are presented. While previous structures were produced from soaking Ca²⁺ into preformed Q138F HincII/DNA crystals, the new structures are derived from cocrystallization with Ca²⁺, Mg²⁺, or Mn²⁺. The Mn²⁺-bound structure provides the first view of a product complex of Q138F HincII with cleaved DNA. Binding studies and a crystal structure show how Ca²⁺ allows trapping of a Q138F HincII complex with noncognate DNA in a catalytically incompetent conformation. Many Q138F HincII/DNA structures show asymmetry, despite the binding of a symmetric substrate by a symmetric enzyme. The various complexes are fit into a model describing the different conformations of the DNA-bound enzyme and show how DNA conformational energetics determine DNA-cleavage rates by the Q138F HincII enzyme.     

Expression,purification, and crystallization of restriction endonuclease PvuII with DNA containing its recognition site

We have overexpressed the type II restriction endonuclease PvuII (R.PvuII) in E. coli, prepared large amounts of the homogeneous enzyme, and crystallized it with an oligonucleotide carrying a PvuII recognition site. The cocrystals are orthorhombic space group P2₁2₁2₁ with cell constants a = 95.8 Å, b = 86.3 Å, c = 48.5 Å, and diffract X-rays to at least 2.7 Å. There is a complex of two protein subunits and one oligonucleotide duplex in the asymmetric unit. © 1994 Wiley-Liss, Inc.