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A Pingoud

Publications and source records attributed to A Pingoud.

At least 73 records · Page 4Linked to original sources
PubMed1996

The production and characterization of artificial heterodimers of the restriction endonuclease EcoRV.

A novel approach to studying the inter- and intrasubunit communication required for the activity of homodimeric proteins is described. It was developed for the restriction endonuclease EcoRV, but should also be useful for other homodimeric enzymes. Two ecorV genes encoding different EcoRV mutants are coexpressed in the same Escherichia coli cell leading to homo- and heterodimeric variants of the enzyme. The two ecorV genes carry either a 5' extension coding for the glutathione-S-transferase or a His6-tag. The EcoRV heterodimer produced in vivo is separated from the two EcoRV homodimers and purified to homogeneity by affinity chromatography. Purified EcoRV heterodimers are stable and are not subject to reassortment of the subunits. To investigate the interdependence of the two catalytic centers, EcoRV heterodimers consisting of one subunit with wild type sequence and one subunit with amino acid substitutions in the PD...(D/E)XK motif, characteristic for the active sites of many restriction endonucleases, were produced. While the homodimeric EcoRV active site mutants are catalytically inactive, the heterodimeric EcoRV variants with one active and one inactive catalytic center display a twofold reduced activity toward oligodeoxynucleotide substrates compared to the wild type, and preferentially nick supercoiled plasmid DNA. From these results we conclude that in the wild type enzyme both catalytic centers function independently of each other.

Biopolymers↗
PubMed1995

Sequence preferences in cleavage of dsDNA and ssDNA by the extracellular Serratia marcescens endonuclease.

The preferred cleavage sites in dsDNA and ssDNA for the extracellular Serratia marcescens endonuclease (commercially available as BENZONASE) were identified by limited digestion of PCR-generated substrates. Two different dsDNA substrates were synthesized by using either radioactively or fluorescent dye labeled primers. ssDNA of identical sequence to one of the fluorescent dye labeled duplex strands was prepared by affinity chromatography. Cleavage experiments carried out under single hit conditions demonstrate that the enzyme shows preferences for GC-rich regions in dsDNA, in particular d(G).d(C)-tracts, and avoids cleavage of d(A).d(T)-tracts. There is a correlation between cleavage at a given position in one strand with cleavage at the same position in the other strand of the duplex. ssDNA cleavage occurs at somewhat different preferred sites than observed in dsDNA. On dsDNA, the Serratia nuclease produces a very different cleavage pattern compared to bovine pancreatic DNase I, with the notable exception that both enzymes avoid d(A).d(T)-tracts. In general, the Serratia nuclease compared to DNase I is a slightly more nonspecific endonuclease that attacks a particular substrate more evenly under standard reaction conditions. At high ionic strength or in the presence of DMSO, it becomes more nonspecific. Addition of urea, however, makes the enzyme more selective than observed under standard conditions. From these results which were confirmed by the results of cleavage experiments with synthetic oligodeoxynucleotides, we conclude that the Serratia nuclease like DNase I is sensitive to global features of the DNA, for example, the width of the minor groove. In addition, localized sequence-dependent interactions between substrate and nuclease determine whether a site is cleaved preferentially. Some of these interactions seem to be the same for ds- and ssDNA.

Base Sequence↗
PubMed1995

Quantitative polymerase chain reaction with enzyme-linked immunosorbent assay detection of selectively digested amplified sample and control DNA.

A quantitative polymerase chain reaction (PCR) method for the exact quantitation of DNA is described that does not require radioactive labeling or electrophoretic separation of product species, thereby avoiding hazardous and time-consuming procedures which have so far impeded routine use of PCR, in particular in the clinical laboratory. Sample and internal control DNA are competitively amplified in a one-tube nested PCR. The control DNA differs from the sample DNA by only two base pairs which change a single restriction site for a new one. Thereby, it is possible to discriminate the two PCR product species by selective restriction enzyme digestion (RED). Because inner PCR primers were labeled with biotin and digoxigenin, respectively, nested PCR products can be immobilized on avidin-coated microtiter plates and quantitated separately by enzyme-linked immunosorbent assay (ELISA) techniques. We demonstrate here that with the combination of selective restriction enzyme digestion and ELISA (RED-ELISA) sample DNA ranging from nanomolar to attomolar concentrations can be quantitated within +/- 10%. This procedure can be easily adapted for quantitation of other PCR products. It is suitable for rapid and automatic screening of many samples in parallel, e.g., for detection and quantitation of pathogens, for quantitation of gene copy numbers or for gene expression after reverse transcription.

Base Sequence↗
PubMed1995

Evidence for an evolutionary relationship among type-II restriction endonucleases.

Type-II restriction-modification (R-M) systems comprise two enzymes, a DNA methyltransferase (MTase) and a restriction endonuclease (ENase), each of which specifically interact with the same 4-8 bp sequence. All type-II MTases share several amino acid (aa) sequence motifs, which makes an evolutionary relatedness among these enzymes probable. The type-II ENases, in contrast, except for some homologous isoschizomers, do not share significant aa sequence similarity. Therefore, ENases in general have been considered unrelated. Here we show that in addition to the analysis of the genotype (aa sequence), a comparison of the phenotype (recognition sequence) of these enzymes can provide independent information regarding evolutionary relationships, and thereby, help to analyze the significance of weak aa sequence similarities. Multistep Monte-Carlo analyses were employed to demonstrate that the recognition sequences of those ENases, which were found to be related by a progressive multiple aa sequence alignment, are more similar to each other than would be expected by chance. This analysis supports the notion that not only type-II MTases, but also type-II ENases did not arise independently in evolution, but rather evolved from one or a few primordial DNA-modifying and DNA-cleaving enzymes, respectively.

Amino Acid Sequence↗
PubMed1995

Evidence for substrate-assisted catalysis in the DNA cleavage of several restriction endonucleases.

Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.

Base Sequence↗
PubMed1995

DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme.

In contrast to many other type II restriction endonucleases, EcoRV binds specifically to DNA only in the presence Mg2+. According to the co-crystal structure of an EcoRV-DNA complex, Mg2+ ion(s) bind to the active site of EcoRV liganded by Glu45, Asp74, and Asp90. Here we present experimental evidence suggesting that the EcoRV-DNA complex also interacts with Mg2+ ions at other sites: (i) We have prepared an EcoRV triple mutant, in which all acidic amino acids in the catalytic center are replaced by alanine. This mutant is catalytically inactive. It binds nonspecifically to DNA in the absence of Mg2+, whereas it binds specifically to DNA in the presence of Mg2+. This means that Mg2+ induces specific DNA binding in this mutant, although all Mg2+ ligands in the catalytic center are removed. Therefore, additional interactions between Mg2+ and the EcoRV-DNA complex probably occur at sites distinct from the catalytic center. (ii) We have measured the specific and nonspecific DNA binding constants of EcoRV and of the triple mutant in the presence and absence of Mg2+. Mg2+ reduces nonspecific binding by 3-4 orders of magnitude, presumably because Mg2+ ions bound to the DNA have to be released upon complex formation. In contrast, the specific binding of the wild-type enzyme and the triple mutant is increased in the presence of Mg2+. This result can only be explained if a Mg2+ ion binds to the specific EcoRV-DNA complex probably at a site distinct from the catalytic center.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1995

A dodecapeptide comprising the extended chain-alpha 4 region of the restriction endonuclease EcoRI specifically binds to the EcoRI recognition site.

The restriction endonuclease EcoRI binds and cleaves DNA containing GAATTC sequences with high specificity. According to the crystal structure, most of the specific contacts of the enzyme to the DNA are formed by the extended chain region and the first turn of alpha-helix alpha 4 (amino acids 137-145). Here, we demonstrate that a dodecapeptide (WDGMAAGNAIER), which is identical in the underlined parts of its sequence to EcoRI amino acids 137-145, specifically binds to GAATTC sequences. The peptide inhibits DNA cleavage by EcoRI but not by BamHI, BclI, EcoRV, HindIII, PacI, and XbaI. DNA cleavage by XbaI is slowed down at sites that partially overlap with EcoRI sites. The peptide inhibits cleavage of GAATTC sites by ApoI, which recognizes the sequence RAATTY. It interferes with DNA methylation by the EcoRI methyltransferase but not by the BamHI methyltransferase. It competes with EcoRI for DNA binding. Based on these results, the DNA binding constant of the peptide to GAATTC sequences was calculated to be 3 x 10(4) M-1. DNA binding is not temperature-dependent, suggesting that binding of the peptide is entropy-driven. As the peptide does not show any nonspecific binding to DNA, its DNA binding specificity is similar to that of EcoRI, in spite of the fact that the affinity is much smaller. These results suggest that contacts to the phosphate groups in EcoRI mainly provide binding affinity, whereas the specificity of EcoRI is based to a large extent on sequence-specific base contacts.

Amino Acid Sequence↗
PubMed1995

Semiautomated quantitative detection of loss of heterozygosity in the tumor suppressor gene p53.

The most frequently altered gene in diverse tumor types is the tumor suppressor gene p53. Typically, normal function is inactivated by point mutation of one allele and deletion of the other. Therefore, loss of heterozygosity (LOH) of intragenic polymorphic markers is a strong indication for p53 involvement in a cancerous lesion. This study shows that a highly polymorphic short tandem repeat (STR) within intron 1 of p53 is an excellent marker for quantitative evaluation of LOH in tumor samples, whose multicolor, fluorescently tagged PCR products are analyzed and quantitated on an automated DNA sequencer. The range of error was analyzed in detail. Discrete allelic profiles were obtained following amplification of DNA from microdissected cell samples of patients with urogenital tumors. By calculating qLOH, the relative allele ratio of a tumor compared with healthy tissue, a quantitative expression for the LOH is obtained. PCR-based tumor DNA typing using fluorescent STR primers and automated analysis provides an enhanced level of accuracy and sensitivity required for routine analysis in clinical practice, where large numbers of tumor samples have to be processed.

Alleles↗
PubMed1994

Protein engineering of the restriction endonuclease EcoRV: replacement of an amino acid residue in the DNA binding site leads to an altered selectivity towards unmodified and modified substrates.

According to the crystal structure analysis of a specific EcoRV/DNA complex, the thymine residues of the recognition sequence -GATATC- are not in direct contact with any amino acid residue of the protein. However, several amino acid residues are sufficiently close that it seemed worthwhile trying to create variants of EcoRV with altered specificity by site-directed mutagenesis. Guided by molecular modelling we have replaced. Asn-188 in the catalytic center of EcoRV by Gln to produce a mutant with a relative preference (compared to wild type EcoRV) for substrates in which one thymine of the recognition sequence is replaced by uracil. We have purified and characterized the resulting N188Q mutant. The selectivity value for the engineered enzyme (the ratio of the kcat/KM values for -GATAUC- versus -GATATC-) differs from that of the wild type enzyme by a factor of more than 200.

Asparagine↗
PubMed1994

Pausing of the restriction endonuclease EcoRI during linear diffusion on DNA.

Linear diffusion is a mechanism to accelerate association rates beyond their three-dimensional diffusional limit. It is employed by the restriction endonuclease EcoRI as well as many other proteins interacting with specific DNA sequences to locate their target sites on the macromolecular substrate. In order to investigate biochemical and biophysical details of the linear diffusion process, we have developed a competitive cleavage assay which allows us to assess with great accuracy the influence of sequence, sequence context, and other structural features on the linear diffusion of EcoRI on DNA. We show here that linear diffusion is not a hopping but a sliding movement in which EcoRI follows the helical pitch of the DNA, because it does not "overlook" any cleavage site. Linear diffusion is slowed when EcoRI encounters sites on the DNA which resemble its recognition site ("star" sites). Pauses of up to 20 s are induced, depending on sequence and orientation of the star site. These data suggest that EcoRI can bind to DNA in two binding modes: one tight, specific, and immobile, leading to DNA cleavage, and another one loose and nonspecific, allowing for linear diffusion. Depending on the similarity between the recognition sequence and the DNA sequence being encountered by EcoRI, there will be a continuous transition between these binding modes. Other proteins bound to the DNA and irregular DNA structures such as bent DNA or a triple helix constitute a barrier that cannot easily be passed by EcoRI.

Base Sequence↗
PubMed1994

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).

Amino Acid Sequence↗
PubMed1994

A procedure for renaturation and purification of the extracellular Serratia marcescens nuclease from genetically engineered Escherichia coli.

Overproduction of the extracellular Serratia marcescens nuclease in Escherichia coli results in aggregation and sequestration of a large amount of the protein in inclusion bodies. Only a relatively small amount is secreted into the medium from which it can be purified following established procedures. The cell-associated insoluble protein can be solubilized in 6 M urea after breaking up the cells by sonication. Renaturation is achieved by dilution or dialysis. Subsequent phosphocellulose chromatography yields a homogeneous protein preparation which is shown by a variety of biochemical and biophysical analyses to be indistinguishable from conventionally prepared material. The high yield (> 10 mg/500-ml culture) and the ease of preparation (2 to 3 days) make this an attractive alternative to previously described procedures.

Amino Acid Sequence↗
PubMed1994

Comparison of N-terminal affinity fusion domains: effect on expression level and product heterogeneity of recombinant restriction endonuclease EcoRV.

The influence of different N-terminal affinity fusion domains on the product heterogeneity of recombinant proteins expressed in Escherichia coli was investigated. N-Terminal extended forms of the restriction endonuclease EcoRV with either glutathione-S-transferase [GST], histidine hexapeptide [(His)6], or a combination of GST and (His)6 [GST-(His)6] were compared to native EcoRV with respect to expression level, susceptibility to inclusion body formation and protein fragmentation. Fingerprinting of product heterogeneity was done by using two-dimensional (2-D) non-equilibrium pH-gradient electrophoresis with subsequent immunoblotting. Fusion proteins containing GST were poorly expressed compared to native EcoRV. In addition, GST fusion proteins were highly susceptible to in-vivo aggregation and fragmentation and displayed more heterogeneity on 2-D immunoblots. However, the sole presence of oligohistidine at the N-terminus of EcoRV proved to be advantageous. Fragmentation of (His)6-EcoRV was not observed and 2-D immunoblots did not show heterogeneous forms of the recombinant protein. In addition, fusion of the histidine-hexapeptide to the N-terminus of native EcoRV increased the expression level of the recombinant protein twofold compared to native EcoRV. Inclusion body formation of the (His)6-EcoRV fusion protein was intensive when cells were grown at 37 degrees C but not at 30 degrees C. The advantage of oligohistidine fusion to EcoRV was finally demonstrated by purifying soluble (His)6-EcoRV in a single-step procedure from crude cell lysates using immobilized metal chelate affinity chromatography.

Biotechnology↗
PubMed1993

Quantitative polymerase chain reaction with oligodeoxynucleotide ligation assay/enzyme-linked immunosorbent assay detection.

Quantitation of nucleic acids by the polymerase chain reaction (PCR) requires coamplification of a control nucleic acid, usually a variant of the sequence to be analyzed, which is added to the sample DNA and amplified in competition. Following PCR, amplified sample and control DNAs are separated by electrophoresis and quantitated, for example by measuring the radioactivity incorporated into the products during the PCR. The need for both electrophoretic separation and radioactive labeling has considerably impeded the use of PCR for routine purposes, e.g., in the clinical laboratory, which requires automatic processing of many samples in parallel. We describe here a quantitative PCR procedure which circumvents electrophoretic separation and detection by radioactivity. It uses a point mutated version of the sample DNA as an internal control. After competitive PCR, amplified sample and control DNA are distinguished by an oligodeoxynucleotide ligation assay (OLA) (Landegren et al., Science 241, 1077-1080, 1988) using two oligodeoxynucleotides, one carrying a biotin-group at the 5'-end and another one with either a digoxigenin (specific for sample DNA) or fluorescein moiety (specific for control DNA), respectively, incorporated close to the 3'-end. Biotinylated oligodeoxynucleotides, educts as well as products of the ligation reaction, are immobilized on avidin-coated microtiter plates. Quantitation of digoxigenin and fluorescein-labeled oligodeoxynucleotide ligation products is achieved by an enzyme-linked immunosorbent assay. This method is very well suited for fast automated or semiautomated PCR.

Base Sequence↗
PubMed1993

Substrate-assisted catalysis in the cleavage of DNA by the EcoRI and EcoRV restriction enzymes.

The crystal structure analyses of the EcoRI-DNA and EcoRV-DNA complexes do not provide clear suggestions as to which amino acid residues are responsible for the activation of water to carry out the DNA cleavage. Based on molecular modeling, we have proposed recently that the attacking water molecule is activated by the negatively charged pro-Rp phosphoryl oxygen of the phosphate group 3' to the scissile phosphodiester bond. We now present experimental evidence to support this proposal. (i) Oligodeoxynucleotide substrates lacking this phosphate group in one strand are cleaved only in the other strand. (ii) Oligodeoxynucleotide substrates carrying an H-phosphonate substitution at this position in both strands and, therefore, lacking a negatively charged oxygen at this position are cleaved at least four orders of magnitude more slowly than the unmodified substrate. These results are supported by other modification studies: oligodeoxynucleotide substrates with a phosphorothioate substitution at this position in both strands are cleaved only if the negatively charged sulfur is in the RP configuration as shown for EcoRI [Koziolkiewicz, M. & Stec, W.J. (1992) Biochemistry 31, 9460-9466] and EcoRV (B. A. Connolly, personal communication). As the phosphate residue 3' to the scissile phosphodiester bond is not needed for strong DNA binding by both enzymes, these findings strongly suggest that this phosphate group plays an active role during catalysis. This proposal, furthermore, gives a straightforward explanation of why in the EcoRI-DNA and EcoRV-DNA complexes the DNA is distorted differently, but in each case the 3' phosphate group closely approaches the phosphate group that is attacked. Finally, an alternative mechanism for DNA cleavage involving two metal ions is unlikely in the light of our finding that both EcoRI and EcoRV need only one Mg2+ per active site for cleavage.

Amino Acid Sequence↗
PubMed1993

Determination of the DNA bend angle induced by the restriction endonuclease EcoRV in the presence of Mg2+.

We have used the method of Zinkel and Crothers (Zinkel, S.S., and Crothers, D.M. (1990) Biopolymers 29, 29-38) to determine the degree of bending induced by the binding of the restriction endonuclease EcoRV to its recognition sequence (-GATATC-). A set of four calibration DNA fragments was constructed that contained zero, two, four, or six phased A-tracts in their centers and an EcoRV site at the 5'-end to account for the electrophoretic influence of the bound protein. The mobilities of these calibration molecules complexed with EcoRV were compared to that of a test DNA containing a central EcoRV site also complexed with EcoRV. The EcoRV-induced bend angle was found to be 44 degrees +/- 4 degrees. These experiments were performed with a catalytically inactive EcoRV mutant that still binds DNA specifically in the presence of Mg2+. In the absence of Mg2+, which is necessary for specific binding, there is no difference in the mobilities of the fragments with a peripheral or a central EcoRV site complexed with EcoRV, indicating that nonspecific binding on average does not lead to measurable DNA bending.

Base Sequence↗
PubMed1993

Mutational analysis of the function of Gln115 in the EcoRI restriction endonuclease, a critical amino acid for recognition of the inner thymidine residue in the sequence -GAATTC- and for coupling specific DNA binding to catalysis.

The Gln115 residue of the EcoRI restriction endonuclease has been proposed to form a hydrophobic contact to the methyl group of the inner thymidine of the EcoRI recognition sequence -GAATTC- and to be involved in intramolecular hydrogen bonds to the mainchain at positions 140 and 143 as well as to the side-chain of Asn173. We have exchanged Gln115 for Ala and Glu by site-directed mutagenesis and analysed the purified mutant proteins (Q115A and Q115E) biochemically and physico-chemically. Q115A and Q115E have the same secondary structure composition as wild-type EcoRI but are less stable towards thermal denaturation than the wild-type enzyme. In contrast to wild-type EcoRI the mutant proteins show a biphasic denaturation profile under alkaline pH, presumably because the amino acid exchange labilizes one part of the molecule, which unfolds before the rest of the protein is denatured. Q115A is catalytically inactive under normal buffer conditions, in part due to a diminished affinity towards DNA. At low ionic strength and alkaline pH, as well as in the presence of Mn2+, i.e. under conditions where wild-type EcoRI shows a relaxed specificity, Q115A is active, however not as much as wild-type EcoRI. Under these conditions it cleaves the canonical sequence -GAATTC- with the same kcat/Km value as the sequence -GAAUTC-, which differs from the former sequence by a single methyl group, while wild-type EcoRI shows a tenfold lower kcat/Km for cleavage of -GAAUTC- than for -GAATTC-. Binding experiments, carried out in the absence of Mg2+, demonstrate that Q115A has a similar affinity towards -GAATTC- as to -GAAUTC-, while wild-type EcoRI binds to -GAATTC- with a tenfold preference over -GAAUTC-. On the basis of these thermodynamic and kinetic results it can be concluded that the hydrophobic contact between the gamma-methylene group of Gln115 and the methyl group of the inner thymidine contributes about 3 kJ/mol (0.7 kcal/mol) to the energy of interaction, both in the ground and the transition state. Q115E is catalytically inactive under normal buffer conditions, but becomes active at low ionic strength or in the presence of Mn2+. Different from Q115A, Q115E is inactive at alkaline pH and its DNA binding affinity is highest at acidic pH.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗