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A Pingoud

Publications and source records attributed to A Pingoud.

At least 19 recordsLinked to original sources
PubMed2005

Type II restriction endonucleases: structure and mechanism.

Type II restriction endonucleases are components of restriction modification systems that protect bacteria and archaea against invading foreign DNA. Most are homodimeric or tetrameric enzymes that cleave DNA at defined sites of 4-8 bp in length and require Mg2+ ions for catalysis. They differ in the details of the recognition process and the mode of cleavage, indicators that these enzymes are more diverse than originally thought. Still, most of them have a similar structural core and seem to share a common mechanism of DNA cleavage, suggesting that they evolved from a common ancestor. Only a few restriction endonucleases discovered thus far do not belong to the PD...D/ExK family of enzymes, but rather have active sites typical of other endonuclease families. The present review deals with new developments in the field of Type II restriction endonucleases. One of the more interesting aspects is the increasing awareness of the diversity of Type II restriction enzymes. Nevertheless, structural studies summarized herein deal with the more common subtypes. A major emphasis of this review will be on target site location and the mechanism of catalysis, two problems currently being addressed in the literature.

Amino Acid Sequence↗
PubMed2005

Affinity modification of the restriction endonuclease SsoII by 2'-aldehyde-containing double stranded DNAs.

Properties of 2'-aldehyde-containing double stranded DNAs (dsDNAs) have been studied for the first time as substrate analogs of the restriction endonuclease SsoII. These reactive oligonucleotides were successfully cross-linked to the restriction endonuclease SsoII by reductive amination, and conditions for DNA-protein conjugate trypsinolysis followed by the oligonucleotide-peptide conjugate purification were optimized. Use of MALDI-TOF mass spectrometry revealed that covalent linkage forms between the sugar moiety of the central pyrimidine nucleoside of the SsoII recognition site and Lys173 of the enzyme. The latter is probably involved in initial steps of enzyme-substrate recognition during dsDNA readout.

Aldehydes↗
PubMed2001

Identification of functionally relevant histidine residues in the apoptotic nuclease CAD.

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD.

Amino Acid Sequence↗
PubMed2001

Structure and function of type II restriction endonucleases.

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.

Base Sequence↗
PubMed2001

Nucleic acid quantification by delta pH measurement.

Nucleic acids are quantitated by UV absorbance measurement, fluorimetry, or hybridization. While the latter method is time-consuming and requires exact knowledge of the sequence, spectroscopic methods require that the sample does not contain UV-absorbing or fluorescent material. An enzymatic method is the measurement of the hyperchromic change upon cleavage of the nucleic acids by nucleases (Kunitz assay). A variation of this assay makes use of the acidification of the solution upon cleavage. We demonstrate here that microgram nucleic acid quantities can be determined when one employs highly active nonspecific nucleases in conjunction with an instrumental setup consisting of a temperature-controlled mixing chamber and miniaturized pH electrodes. Because this method determines the total amount of phosphodiester bonds cleaved, it is independent of the composition or the secondary structure of the nucleic acid and, under certain precautions, represents a simple and robust alternative to optical assays for the determination of either the total nucleic acid concentration or the activity of nucleases in biochemical samples.

Anabaena↗
PubMed2001

Immobilization of sugar-non-specific nucleases by utilizing the streptavidin--biotin interaction.

Due to their high enzymatic activity, the sugar-non-specific endonucleases from Serratia marcescens and Anabaena can be used for a number of applications, such as the removal of contaminating genetic material from biological preparations, footprinting studies, and the determination of nucleic acids in biochemical samples. These methods would benefit from immobilized nucleases. For this purpose, a single cysteine residue was added at the N-terminus of the Serratia and Anabaena nucleases and subsequently modified with a maleimide-biotin conjugate. Alternatively, a biotin acceptor domain was fused to the Anabaena nuclease, allowing biotinylation during expression in E. coli without a further chemical step. The attachment of biotin-modified nucleases to streptavidin-coated paramagnetic beads and to streptavidin-coated surface plasmon resonance sensor chips (to study interactions with substrate and inhibitor) worked well when aggregates present in the protein preparations were removed by ultrafiltration. These methods should be of general use for similar enzyme systems.

Amino Acid Sequence↗
PubMed2001

Sau3AI, a monomeric type II restriction endonuclease that dimerizes on the DNA and thereby induces DNA loops.

Here, we report that Sau3AI, an unusually large type II restriction enzyme with sequence homology to the mismatch repair protein MutH, is a monomeric enzyme as shown by gel filtration and ultracentrifugation. Structural similarities in the N- and C-terminal halves of the protein suggest that Sau3AI is a pseudo-dimer, i.e. a polypeptide with two similar domains. Since Sau3AI displays a nonlinear dependence of cleavage activity on enzyme concentration and a strong preference for substrates with two recognition sites over those with only one, it is likely that the functionally active form of Sau3AI is a dimer of a pseudo-dimer. Indeed, electron microscopy studies demonstrate that two distant recognition sites are brought together through DNA looping induced by the simultaneous binding of two Sau3AI molecules to the DNA. We suggest that the dimeric form of Sau3AI supplies two DNA-binding sites, one that is associated with the catalytic center and one that serves as an effector site.

Amino Acid Sequence↗
PubMed2001

Influence of fluorophor dye labels on the migration behavior of polymerase chain reaction--amplified short tandem repeats during denaturing capillary electrophoresis.

The determination of the length of polymerase chain reaction (PCR)-amplified short tandem repeats (STRs) by denaturing capillary electrophoresis (CE) is a standard procedure for purposes of genotyping. We show that dye-specific mobility anomalies exist for 5'-fluorophor-labelled single-stranded DNA (ssDNA) fragments in CE using the performance-optimized polymer 4 (POP4) buffer sieving matrix, containing the entangled poly(N,N-dimethylacrylamide) polymer, urea, and 2-pyrrolidinone. The dye-specific retardation effects relative to coseparated GeneScan-500 [TAMRA] standard fragments can lead to wrong genotyping, even for allele-specific fragments of pentanucleotide STRs, when comparing the relative calculated sizes of identical fragments, labelled with rhodamine (ROX, TAMRA) or fluorescein dyes (FAM, 6-FAM, HEX, JOE, NED, TET): The size of fluorescein dye-labelled fragments of appr. 100 b in length appears to be smaller by up to 6.5 b. This effect becomes more dramatic with decreasing size: a 6-FAM-labelled 24-mer oligonucleotide appeared to be smaller by 11 b. In contrast, in classical urea/polyacrylamide slab-gel electrophoresis only a small dye-specific retardation of identical fragments is observed. The dye-specific effects are superimposed by weaker size and sequence-dependent anomalies of fragment mobility. Therefore, in denaturing CE the coseparation of a defined allele ladder labelled with the same dye as the unknown sample fragments remains the method of choice for accurate genotyping.

DNA, Single-Stranded↗
PubMed2001

Comparison between Taq DNA polymerase and its Stoffel fragment for quantitative real-time PCR with hybridization probes.

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.

DNA-Directed DNA Polymerase↗
PubMed2000

Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI.

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.

Amino Acid Substitution↗
PubMed2000

Genetic engineering of Escherichia coli to produce a 1:1 complex of the anabaena sp. PCC 7120 nuclease NucA and its inhibitor NuiA.

A series of T7-promoter based bicistronic expression vectors was constructed in order to produce the complex of the Anabaena sp. PCC 7120 DNA/RNA non-specific nuclease NucA and its inhibitor NuiA. With all constructs, tandem expression of nucA and nuiA results in aggregation and inclusion body formation of NucA, independent of the order of the genes, the relative expression of the two proteins and the temperature applied during expression. Two constructs in which nuiA is the first and nucA the second cistron lead to an approximately one order of magnitude higher expression of nuiA compared with nucA. In these cells inclusion bodies are formed which contain NucA and NuiA in a 1:1 molar ratio. The complex can be solubilized with 6M urea after disruption of the cells by sonication, renatured by dialysis and purified to homogeneity. 2mg of the complex are obtained from 1l Escherichia coli culture. As shown by gel filtration and analytical ultracentrifugation, our system leads to a highly pure and homogeneous complex preparation, as required for biophysical and structural studies. Thus, our new method is a superior alternative for the production of the NucA/NuiA complex in which separately produced nuclease and inhibitor are mixed, and an excess of one or the other component, as well as aggregates of NucA, have to be removed from the preparation.

Algal Proteins↗
PubMed2000

A model for the PI-SceIxDNA complex based on multiple base and phosphate backbone-specific photocross-links.

We have synthesized different oligodeoxynucleotides carrying, in single positions of the >36 bp recognition site of PI-SceI, photoreactive base analogues (5-iododeoxypyrimidines) or phosphate modifications (p-azidophenacylphosphorothioates) and used them in photocross-linking experiments with PI-SceI to probe the protein-DNA interface of the specific complex between the homing endonuclease PI-SceI and its DNA substrate. One base-specific and several backbone-specific cross-links were analyzed in detail: the cross-linking positions were identified by Edman degradation of isolated cross-linked peptidexoligodeoxynucleotide adducts and confirmed by site-directed mutagenesis. Based on these results and the crystal structure of PI-SceI, a model for the structure of the PI-SceIxDNA complex is proposed.

Base Pairing↗
PubMed2000

Mechanism of DNA cleavage by the DNA/RNA-non-specific Anabaena sp. PCC 7120 endonuclease NucA and its inhibition by NuiA.

A structural model of the DNA/RNA non-specific endonuclease NucA from Anabaena sp. PCC7120 that has been obtained on the basis of the three-dimensional structure of the related Serratia nuclease, suggests that the overall architecture of the active site including amino acid residues H124, N155 and E163 (corresponding to H89, N119 and E127 in Serratia nuclease) is similar in both nucleases. Substitution of these residues by alanine leads to a large reduction in activity (<0.1 %), similarly as observed for Serratia nuclease demonstrating that both enzymes share a similar mechanism of catalysis with differences only in detail. NucA is inhibited by its specific polypeptide inhibitor with a K(i) value in the subpicomolar range, while the related Serratia nuclease at nanomolar concentrations is only inhibited at an approximately 1000-fold molar excess of NuiA. The artificial chromophoric substrate deoxythymidine 3',5'-bis-(p-nitrophenyl phosphate) is cleaved by NucA as well as by Serratia nuclease. Cleavage of this analogue by NucA, however, is not inhibited by NuiA, suggesting that small molecules gain access to the active site of NucA in the enzyme-inhibitor complex under conditions where cleavage of DNA substrates is completely inhibited. The active site residue E163 seems to be the main target amino acid for inhibition of NucA by NuiA, but R93, R122 and R167 (corresponding to K55, R87, R131 in Serratia nuclease) are also involved in the NucA/NuiA interaction. NuiA deletion mutants show that the structural integrity of the N and C-terminal region of the inhibitor is important for complex formation with NucA and inhibition of nuclease activity. Based on these results a mechanism of DNA cleavage by NucA and its inhibition by NuiA is proposed.

Amino Acid Sequence↗
PubMed2000

Identification of a base-specific contact between the restriction endonuclease SsoII and its recognition sequence by photocross-linking.

A target sequence-specific DNA binding region of the restriction endonuclease Sso II was identified by photocross-linking with an oligodeoxynucleotide duplex which was substituted with 5-iododeoxy-uridine (5-IdU) at the central position of the Sso II recognition site (CCNGG). For this purpose the Sso II-DNA complex was irradiated with a helium/cadmium laser (325 nm). The cross-linking yield obtained was approximately 50%. In the presence of excess unmodified oligodeoxynucleotide or with oligode-oxynucleotides substituted with 5-IdU elsewhere, no cross-linking was observed, indicating the specificity of the cross-linking reaction. The cross-linked Sso II-oligodeoxynucleotide complex was digested with chymotrypsin, a cross-linked peptide-oligodeoxy-nucleotide complex isolated and the site of cross-linking identified by Edman sequencing to be Trp61. In line with this identification is the finding that the W61A variant cannot be cross-linked with the IdU-substituted oligodeoxynucleotide, shows a decrease in affinity towards DNA and is inactive in cleavage. It is concluded that the region around Trp61 is involved in specific binding of Sso II to its DNA substrate.

Base Pairing↗
PubMed2000

On the possibilities and limitations of rational protein design to expand the specificity of restriction enzymes: a case study employing EcoRV as the target.

The restriction endonuclease EcoRV has been characterized in structural and functional terms in great detail. Based on this detailed information we employed a structure-guided approach to engineer variants of EcoRV that should be able to discriminate between differently flanked EcoRV recognition sites. In crystal structures of EcoRV complexed with d(CGGGATATCCC)(2) and d(AAAGATATCTT)(2), Lys104 and Ala181 closely approach the two base pairs flanking the GATATC recognition site and thus were proposed to be a reasonable starting point for the rational extension of site specificity in EcoRV [Horton,N.C. and Perona,J.J. (1998) J. Biol. Chem., 273, 21721-21729]. To test this proposal, several single (K104R, A181E, A181K) and double mutants of EcoRV (K104R/A181E, K104R/A181K) were generated. A detailed characterization of all variants examined shows that only the substitution of Ala181 by Glu leads to a considerably altered selectivity with both oligodeoxynucleotide and macromolecular DNA substrates, but not the predicted one, as these variants prefer cleavage of a TA flanked site over all other sites, under all conditions tested. The substitution of Lys104 by Arg, in contrast, which appeared to be very promising on the basis of the crystallographic analysis, does not lead to variants which differ very much from the EcoRV wild-type enzyme with respect to the flanking sequence preferences. The K104R/A181E and K104R/A181K double mutants show nearly the same preferences as the A181E and A181K single mutants. We conclude that even for the very well characterized restriction enzyme EcoRV, properties that determine specificity and selectivity are difficult to model on the basis of the available structural information.

Alanine↗
PubMed2000

Automated purification of His6-tagged proteins allows exhaustive screening of libraries generated by random mutagenesis.

In the course of site-directed mutagenesis or directed evolution experiments, large numbers of protein variants are often generated. To characterize functional properties of individual mutant proteins in vitro, a rapid and reliable protein purification system is required. We have developed an automated method for the parallel purification of 96 different protein variants that takes about two hours. Using a 96-well format, the whole process can be performed automatically by a pipetting robot. Coupled with a suitable assay, again using a 96-well format, all variants can be functionally characterized within a few hours. The protein purification procedure described here is based on the interaction between His6-tagged proteins and Ni-NTA-coated microplates. Typical yields are 3-8 pmol purified protein/well, which is sufficient to analyze most enzymatic activities. Using this procedure, we have purified and characterized variants of the restriction endonuclease EcoRV, which were produced in an effort to enhance the selectivity of this enzyme. For this purpose, three amino acid residues were randomized in a region known from the co-crystal structure to be located at the protein-DNA interface. From a library of about 1200 variants, predominantly single and double mutants, more than 1000 variants were purified and characterized in parallel, which corresponds to an almost complete screening of the library.

Automation↗
PubMed2000

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Cyclin-Dependent Kinase Inhibitor p16↗