Type IIE Research Articles

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca2+ increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site.

Type IIE and IIF Restriction Endonucleases Interacting with Two Recognition Sites on DNA

Recently, it was revealed that restriction endonucleases widely used in genetic engineering and molecular biology are diverse not only in DNA sequence specificities but also in mechanisms of their interaction with DNA. In the review type IIE and IIF restriction endonucleases which require the simultaneous interaction with two copies of their recognition sequence for effective hydrolysis of DNA are considered. Crystal structures of these enzymes and their complexes with DNA as well as stepwise interaction with DNA, mechanisms of catalysis and enzyme-mediated DNA looping are discussed. A novel type of DNA-protein recognition was found for type IIE endonucleases when two copies of the same DNA sequence specifically interact with two different amino acid sequences and two structural motifs located in one polypeptide chain.

Crystal Structure of Type IIE Restriction Endonuclease EcoRII Reveals an Autoinhibition Mechanism by a Novel Effector-binding Fold

EcoRII is a type IIE restriction endonuclease that interacts with two copies of the DNA recognition sequence 5′CCWGG, one being the actual target of cleavage, the other serving as the allosteric effector. The mode of enzyme activation by effector binding is unknown. To investigate the molecular basis of activation and cleavage mechanisms by EcoRII, the crystal structure of EcoRII mutant R88A has been solved at 2.1 Å resolution. The EcoRII monomer has two domains linked through a hinge loop. The N-terminal effector-binding domain has a novel DNA recognition fold with a prominent cleft. The C-terminal catalytic domain has a restriction endonuclease-like fold. Structure-based sequence alignment identified the putative catalytic site of EcoRII that is spatially blocked by the N-terminal domain. The structure together with the earlier characterized EcoRII enzyme activity enhancement in the absence of its N-terminal domain reveal an autoinhibition/activation mechanism of enzyme activity mediated by a novel effector-binding fold. This is the first case of autoinhibition, a mechanism described for many transcription factors and signal transducing proteins, of a restriction endonuclease.

A single mutation of restriction endonuclease EcoRII led to a new crystal form that diffracts to 2.1 A resolution.

R88A, a mutant of the type IIE restriction endonuclease EcoRII, has been crystallized in space group P2(1), with unit-cell parameters a = 58.7, b = 92.4, c = 88.3 A, beta = 108.1 degrees. There are two monomers in the asymmetric unit and the solvent content is estimated to be 50% by volume. The crystals diffract to 2.1 A resolution, which is much higher than that of the wild type, which diffracted to 2.8 A resolution. The mutant crystals have been used in the identification of an excellent heavy-atom derivative.

Direct monitoring of EcoRII restriction enzyme reactions on a 27MHz quartz-crystal microbalance

EcoRII is a type IIe restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its recognition site. Gaining insights about reaction property of EcoRII binding to its recognition site and effect of divalent metal ion on DNA cleavage process, a DNA-immobilized quartz crystal microbalance (QCM), which enables real-time monitoring both the binding of enzyme and the cleavage reaction on DNA strands as mass changes, was utilized.

A new crystal form of restriction endonuclease EcoRII that diffracts to 2.8 A resolution.

EcoRII, a type IIe restriction endonuclease, has been crystallized in space group P2(1), with unit-cell parameters a = 58.3, b = 127.8, c = 59.9 A, beta = 91.4 degrees. There are two monomers in the asymmetric unit and the solvent content is estimated to be 49% by volume. The crystals diffract to 2.8 A resolution, which is much higher than that of the previously reported cubic crystal form, which diffracted to 4 A resolution.

Evolutionary Relationship between Different Subgroups of Restriction Endonucleases

The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.

Asymmetric Photocross-linking Pattern of Restriction EndonucleaseEcoRII to the DNA Recognition Sequence

The EcoRII homodimer engages two of its recognition sequences (5'-CCWGG) simultaneously and is therefore a type IIE restriction endonuclease. To identify the amino acids of EcoRII that interact specifically with the recognition sequence, we photocross-linked EcoRII with oligonucleotide substrates that contained only one recognition sequence for EcoRII. In this recognition sequence, we substituted either 5-iododeoxycytidine for each C or 5-iododeoxyuridine for A, G, or T. These iodo-pyrimidine bases were excited using a UV laser to result in covalent cross-linking products. The yield of EcoRII photocross-linked to the 5'-C of the 5'-CCAGG strand of the recognition sequence was 45%. However, we could not photocross-link EcoRII to the 5'-C of the 5'-CCTGG strand. Thus, the contact of EcoRII to the bases of the recognition sequence appears to be asymmetric, unlike that expected for most type II restriction endonucleases. Tryptic digestion of free and of cross-linked EcoRII, followed by high performance liquid chromatography (HPLC) separation of the individual peptides and Edman degradation, identified amino acids 25-49 of EcoRII as the cross-linking peptide. Mutational analysis of the electron-rich amino acids His(36) and Tyr(41) of this peptide indicates that Tyr(41) is the amino acid involved in the cross-link and that it therefore contributes to specific DNA recognition by EcoRII.

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function

Type 2 von Willebrand disease causing defective von Willebrand factor-dependent platelet function comprises mainly subtypes 2A, 2B and 2M. The diagnosis of type 2 von Willebrand disease may be guided by the observation of a disproportionately low level of ristocetin co-factor activity or collagen-binding activity relative to the von Willebrand factor antigen level. The decreased platelet-dependent function is often associated with an absence of high molecular weight multimers (types 2A and 2B), but the high molecular weight multimers may also be present (type 2M and some type 2B), and supranormal multimers may exist (as in the Vicenza variant). Today, the identification of mutations in particular domains of the pre-pro-von Willebrand factor is helpful to classify these variants and to provide further insight into the structure–function relationship and the biosynthesis of von Willebrand factor. Thus, mutations in the D2 domain, involved in the multimerization process, are found in patients with type 2A, formerly named IIC von Willebrand disease. Mutations in the D3 domain characterize the Vicenza variant, or type IIE patients. Mutations in the A1 domain may modify the binding of von Willebrand factor multimers to platelets, either increasing (type 2B) or decreasing (types 2M and 2A/2M) the affinity of von Willebrand factor for platelets. In type 2A disease, molecular abnormalities identified in the A2 domain, which contains a specific proteolytic site, are associated with alterations in folding that impair the secretion of von Willebrand factor or increase its susceptibility to proteolysis. Finally, a mutation localized in the C terminus cysteine knot domain, which is crucial for the dimerization of von Willebrand factor subunit, has been identified in a rare subtype 2A, formerly named IID.

Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase.

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.

Imaging DNA Loops Induced by Restriction Endonuclease EcoRII: A SINGLE AMINO ACID SUBSTITUTION UNCOUPLES TARGET RECOGNITION FROM COOPERATIVE DNA INTERACTION AND CLEAVAGE

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.

関節内転位を伴うしょう骨骨折に対するｐｌａｔｅを用いた観血的治療の経験

Seventeen cases with intra-articular displaced fracture of the calcaneus were treated surgically using the plate. They were classified into type II-C (3 cases), II-E (8 cases), and II-F (6 cases) according to Essex-Lopresti's classification.All cases achieved bone union. Using the criteria for assessment described by Maxfield, excellent results were achieved in 9 cases. very good in 7 and fair in 1.Open reduction and internal fixation is a useful method for treating intra-articular displaced fracture of the calcaneus.

Histochemical and electrophoretic analysis of the primary flight muscle of several phyllostomid bats

We identify a novel histochemical fibre type that is correlated with a unique myosin heavy chain isoform in the pectoralis muscle of the two bats Artibeus lituratus and Carollia perspicillata (Phyllostomidae). Pectoralis muscles sampled from four other species (Phyllostomus hastatus, Platyrrhinus helleri, Glossophaga soricina, and Diaemus youngi) exhibited myosin heavy chain isoforms with electrophoretic properties identical with those of A. lituratus andC. perspicillata. The pectoralis muscles of A. lituratus and C. perspicillata consisted mainly of the newly described type IIe fibre. Diaemus youngi differed from the other species studied by having an FM5 native myosin isoform, a feature shared with Desmodus rotundus. We hypothesize that the two-fibre-type composition of the pectoralis muscle in the phyllostomid bats may be related to a "two-gear" locomotor strategy and represents a synapomorphy. The distribution of fibre types and myosin isoforms correlated with family-level phylogenetic affinity rather than with functional characteristics. One of the fibre types was identical with type IIa of terrestrial mammals and a correlated IIa isoform pattern upon electrophoretic analysis. The IIa fibres had high oxidative and glycolytic potential. The second fibre type, which we call type IIe, had a histochemical and immunocytochemical pattern inconsistent with interpretation as a type IIb muscle fibre. Electrophoretic analysis confirmed that isoforms correlated with IIe fibres migrated at different rates from type I, IIa, IIb, and IIx isoforms. The IIe fibres had a fast oxidative metabolic pattern and were at least twice as numerous as IIa fibres. Thus, a novel type IIe fibre is predominant in the pectoralis of six phyllostomid species that exhibit highly divergent body sizes and (or) flight styles.

Cooperative Binding Properties of Restriction Endonuclease EcoRII with DNA Recognition Sites

EcoRII is a member of the expanding group of type IIe restriction endonucleases that share the distinguishing feature of requiring cooperativity between two recognition sites in their substrate DNA. To determine the stoichiometry of the active DNA-enzyme complex and the mode of cooperative interaction, we have investigated the dependence of EcoRII cleavage on the concentration of EcoRII dimers. Maximal restriction was observed at dimer/site ratios of 0.25 and 0. 5. The molecular weight of the DNA-enzyme complex eluted from a gel filtration column also corresponds to a dimeric enzyme structure bound to two substrate sites. We conclude that one EcoRII dimer is sufficient to interact cooperatively with two DNA recognition sites. A Lac repressor "barrier" bound between two normally reactive EcoRII sites did not inhibit restriction endonuclease activity, indicating that cooperativity between EcoRII sites is achieved by bending or looping of the intervening DNA stretch. Comparative cleavage of linear substrates with differently spaced interacting sites revealed an inverse correlation between cleavage rate and site distance. At the optimal distance of one helical turn, EcoRII cleavage is independent of the orientation of the recognition sequence in the DNA double strand.

Application of oligonucleotide activation to restriction endonuclease NarI.

Restriction endonuclease NarI cleaves DNA using a two-site mechanism, placing it in the Type IIe class of restriction endonucleases. Although these enzymes have very useful recognition sequences, the two-site mechanism limits the practical application. Site preferences often cause incomplete substrate digestion. Oligonucleotide activation of NarI eliminates incomplete digestions, making it possible to use NarI restriction sites in many common molecular biology techniques. A modified oligonucleotide was chosen for optimal activation of restriction endonuclease NarI. This oligonucleotide was demonstrated to allow complete digestion in many commonly used substrates.

Collagen markers during anticancer therapy for lung cancer

Recently, many studies have reported that collagen metabolism markers, such as the aminoterminal propeptide of type IIE collagen (PIIIP) and the 7-S domain of type TV collagen (7-S collagen), increased in malignancy and that, they correlate with the clinical behavior of the disease. The objective of the present study was to assess the serum concentration of these collagen markers in relation to the clinical progression of the disease and during chemotherapy in lung cancer patients. This study comprised 48 lung cancer patients. Sixteen patients underwent cisplatin-based chemotherapy. Samples taken from controls were available for comparison. Serum concentrations of PIIIP and 7S-collagen were significantly higher as compared to the control group. While the collagen markes tended to decrease in the responder patients, they increased significantly after chemotherapy in the non-responder group. Collagen markers are suggested to be potentially useful for the early evaluation of the tumor response to chemotherapy in lung cancer.

Histochemical and myosin composition of vampire bat (Desmodus rotundus) pectoralis muscle targets a unique locomotory niche.

The vampire bat pectoralis muscle contains at least four fiber types distributed in a nonhomogeneous pattern. One of these fiber types, here termed IIe, can be elucidated only by adenosine triphosphatase (ATPase) histochemistry combined with reactions against antifast and antislow myosin antibodies. The histochemical and immunohistochemical observations indicate a well-developed specialization of function within specific regions of the muscle. In parallel, analyses of native myosin isoforms and myosin heavy chain isoforms indicate two points. First, the histochemical "type IIe" fiber is predominant in cranial portions of the muscle, and myosin extracted from these regions exhibits a unique electrophoretic mobility not observed in the myosin isoforms of more traditional laboratory mammals. Second, the type I fibers are confined to the pectoralis abdominalis muscle and a small adjacent region of the caudal part of the pectoralis. This pattern of type I fiber distribution is considered a derived character state compared to muscle histochemical phenotype and isoform composition in the pectoralis muscles of other phyllostomids we have studied (Artibeus jamaicensis, Artibeus lituratus, Carollia perspicillata). We relate this to the unique locomotory needs of the common vampire bat, Desmodus rotundus.

Subunit composition of plasma von Willebrand factor. Cleavage is present in normal individuals, increased in IIA and IIB von Willebrand disease, but minimal in variants with aberrant structure of individual oligomers (types IIC, IID, and IIE).

We have evaluated the subunit composition of plasma von Willebrand factor (vWF) and found evidence that cleavage is present in normal individuals, increased in IIA and IIB von Willebrand disease (vWD), but decreased or absent in variants with aberrant structure of individual oligomers. vWF was rapidly purified from plasma on an analytical scale by monoclonal antibody immunoaffinity chromatography in the presence of protease inhibitors. After reduction and electrophoresis in 5% polyacrylamide gels containing sodium dodecyl sulfate, fragments of 189, 176, and 140 kD, as well as the predominant 225-kD subunit, were identified in plasma vWF from 25 normal individuals. The vWF polypeptides were detected by immunoblotting with a mixture of 55 anti-vWF monoclonal antibodies followed by 125I-rabbit anti-mouse antibody and autoradiography. In five individuals with type IIA and five individuals with type IIB vWD, the proportions of 176 and 140-kD fragments were increased relative to the intact 225-kD subunit, as determined by excising each band and quantitating incorporated radioactivity. In contrast, these fragments were either not detectable or were present in only trace amounts in variants with abnormal structure of individual oligomers (types IIC and IID, and a new variant, type IIE vWD). The results reported here provide evidence that absence of large vWF multimers in these two groups of variants results from different mechanisms. In addition, they demonstrate that partial cleavage of the plasma vWF subunit is a normal event.

ChemInform Abstract: QUANTITATIVE STRUCTURE‐ACTIVITY RELATIONSHIP OF DOUBLE ALKYL CHAIN DRUGS

The quantitative structure--activity relationship of double alkyl chain drugs, including alkanols, aliphatic esters, ketones, barbiturates, amphetamines, butyrylcholinesterase inhibitors, antimalarials, and rifamycin amides, is investigated. A series of double-chain homologues, CnH2n+1XCmH2m+1, in which n changes, keeping m constant, is classified into three types: in type IIL, n greater than m; in type IIE, n = m; in type IIS, n less than m. When a linear relationship, vis., log (1/C) = an + b, holds, the slope a depends on the type; aI greater than or equal to aIIL greater than aIIE greater than aIIS. Here aI means the slope for single-chain homologues. The same order is observed for the equation, log hydrophobicity = an + b, where the hydrophobicity of drug denotes the water solubility, the critical micelle concentration, and the partition coefficient for the 1-octanol--water phases. Therefore, decreased biological activity of a double-chain drug relative to that of a single-chain isomer can be explained by a decreased hydrophobicity of the double-chain drug, due to the intramolecular association of these chains in water. When a parabolic relationship between log (1/C) and n holds, the optimum n depends on the type: nopI less than nopIIL less than nopIIE. This order is also explicable on the basis of a decreased hydrophobicity of double-chain drug. The N-dealklation rate of amphetamines in vivo appears to be affected by the steric factor as well as the hydrophobic factor. A decreased hydrophobicity of double-chain compounds should be taken into consideration for estimating their partition coefficients.

Quantitative structure--activity relationship of double alkyl chain drugs.

The quantitative structure--activity relationship of double alkyl chain drugs, including alkanols, aliphatic esters, ketones, barbiturates, amphetamines, butyrylcholinesterase inhibitors, antimalarials, and rifamycin amides, is investigated. A series of double-chain homologues, CnH2n+1XCmH2m+1, in which n changes, keeping m constant, is classified into three types: in type IIL, n greater than m; in type IIE, n = m; in type IIS, n less than m. When a linear relationship, vis., log (1/C) = an + b, holds, the slope a depends on the type; aI greater than or equal to aIIL greater than aIIE greater than aIIS. Here aI means the slope for single-chain homologues. The same order is observed for the equation, log hydrophobicity = an + b, where the hydrophobicity of drug denotes the water solubility, the critical micelle concentration, and the partition coefficient for the 1-octanol--water phases. Therefore, decreased biological activity of a double-chain drug relative to that of a single-chain isomer can be explained by a decreased hydrophobicity of the double-chain drug, due to the intramolecular association of these chains in water. When a parabolic relationship between log (1/C) and n holds, the optimum n depends on the type: nopI less than nopIIL less than nopIIE. This order is also explicable on the basis of a decreased hydrophobicity of double-chain drug. The N-dealklation rate of amphetamines in vivo appears to be affected by the steric factor as well as the hydrophobic factor. A decreased hydrophobicity of double-chain compounds should be taken into consideration for estimating their partition coefficients.