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Biomedical subjects

D Kupper

Publications and source records attributed to D Kupper.

12 recordsLinked to original sources
PubMed2000

A fluorescence based non-radioactive electrophoretic mobility shift assay.

Electrophoretic mobility shift assay (EMSA) or gel shift assay is one of the most powerful methods for studying protein-DNA interactions. Typically, 32P-labeled DNA probes containing the sequence bound by the protein of interest are used in EMSA (rEMSA). Although rEMSA is sensitive and practicable, it relies on the handling of hazardous radioisotopes, and does not easily allow quantification. We developed a non-radioactive procedure using fluorescence (Cyano dye Cy5) labeled oligodeoxynucleotide duplexes as specific probes (fEMSA) and an automatic DNA sequencer for analysis. Testing different DNA-binding proteins (restriction endonuclease EcoRII, transcription factor NFkappaB and it's subunit p50) the results in fEMSA and rEMSA are similar in regard to quality, reproducibility, and sensitivity. fEMSA allows a semiquantitative screening of large amounts of samples for specific DNA binding activities and is, therefore, a high throughput technology for semiquantitative analysis of DNA-protein interaction.

Carbocyanines↗
PubMed1999

Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires.

Target sequence-specific DNA binding regions of the restriction endonuclease EcoRII were identified by screening a membrane-bound EcoRII-derived peptide scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex. Dodecapeptides overlapping by nine amino acids and representing the complete protein were prepared by spot synthesis. Two separate DNA binding regions, amino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by exchanging every residue of both binding sites for all other amino acids demonstrated that replacing basic residues in the consensus motifs significantly reduced DNA binding. EcoRII mutant enzymes generated by substituting alanine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structure was unaffected. Furthermore, Glu96, which is part of a potential catalytic motif and also locates to DNA binding site I, was demonstrated to be critical for DNA cleavage and binding. Homology studies of DNA binding site II revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionally related DNA binding sites in diverse restriction endonucleases with recognition sequences containing terminal C:G or G:C pairs.

Amino Acid Sequence↗
PubMed1998

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.

Bacterial Proteins↗
PubMed1995

Restriction endonucleases functionally interacting with two DNA sites.

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

Binding Sites↗
PubMed1995

Overproduction of His-tagged EcoRII restriction endonuclease and terminally deleted mutant proteins.

EcoRII was the first restriction endonuclease (ENase) reported requiring the cooperative interaction with at least two DNA sites for activity. Using two different expression systems the enzyme could be purified and its special substrate requirements were further analyzed. At the present state of knowledge we suggest a model of simultaneous binding of two DNA sites to one dimeric enzyme molecule (see Krüger et al., FEMS Microbiol. Rev. (1995) in press).

Binding Sites↗
PubMed1995

Hyperexpressed EcoRII renatured from inclusion bodies and native enzyme both exhibit essential cooperativity with two DNA sites.

EcoRII was the first restriction endonuclease (ENase) reported needing the cooperative interaction with at least two DNA sites for activity. We constructed an EcoRII-overproducing strain of Escherichia coli by placing the coding sequence under control of the T7 gene 10 regulatory elements. The yield of EcoRII expression could be increased to about 10% of total soluble cellular protein. Inclusion bodies are formed that mainly consist of insoluble EcoRII molecules. After solubilization by 6 M guanidine hydrochloride refolding of the enzyme was achieved by dilution into appropriate buffer. The endonuclease was purified to homogeneity from both the soluble protein fraction and the protein renatured from inclusion bodies. Their identity was proven by circular dichroism and analysis of enzyme activity with respect to the special substrate requirements of EcoRII. It is shown that EcoRII cleavage of oligodeoxyribonucleotide duplexes (oligo duplexes) with only one recognition site follows a sigmoidal concentration dependence, i.e., they cannot be cleaved below a distinct low DNA concentration where simultaneous interaction with two substrate molecules is no longer possible. We demonstrate that the restriction of oligo duplexes containing two recognition sites does not show this concentration dependence, confirming an intramolecular site cooperativity.

Base Sequence↗
PubMed1995

The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes.

Studies on phage T3 and T7 have shown that these viruses avoid restriction not only by the phage-coded Ocr (and S-adenosylmethionine hydrolase) protein functions or by the complete loss of specific recognition sites for certain restriction endonucleases from their genomes, but also that there are two additional modes: resistance towards EcoP15 (which recognizes a non-symmetrical sequence) is achieved by an identical orientation of all the recognition sites in the virus genome (strand bias) and in the case of EcoRII by the extreme reduction in number and thereby greater distance between recognition sites in the genome. These observations led to the discovery that certain restriction endonucleases require the simultaneous cooperation with two DNA sites for their function, as well as to the ongoing elucidation of the molecular modes of action of these enzymes. Type II and type III enzymes display fundamentally different mechanisms of protein-DNA interaction. For EcoRII we favor a model of simultaneous binding of two DNA sites to a dimeric enzyme molecule (neighbouring sites of the same, looping, DNA molecule or sites located on different DNA molecules), while the action of EcoP15 seems to conform with a tracking-collision model of two enzyme molecules bound to inversely oriented recognition sites. In addition to podoviruses T3 and T7, strand bias of recognition sequences for different type III DNA modification-restriction enzymes is also observed in the inoviruses M13, IKE and PF3.

Bacteriophage T3↗
PubMed1993

Use of specific oligonucleotide duplexes to stimulate cleavage of refractory DNA sites by restriction endonucleases.

There are numerous restriction endonucleases (ENases) which are known never to achieve total cleavage of certain unmethylated target DNAs. In addition to EcoRII we found seven ENases (AtuBI, Cfr9I, Eco57I, Ksp632I, NaeI, NarI, and SauBMKI) that were stimulated by oligodeoxyribonucleotide (oligo) duplexes containing enzyme-specific recognition sequences to cut the target DNAs much more efficiently and in most cases even to completion. These enzymes are class-II and class-IIS Enases isolated from different bacterial species and possess a varying number of specific sites in the refractory DNA substrates. For DNA analysis and large-scale preparation of certain restriction fragments where complete digestions are essential we recommend taking into account the fact that various ENases can be activated by specific oligo duplexes to drive restriction digestions to completion.

Base Sequence↗
PubMed1989

Cloning and structure of the BepI modification methylase.

The gene coding for a CGCG specific DNA methylase has been cloned in E. coli from Brevibacterium epidermidis. The enzyme, named BepI methylase, is probably the cognate methylase of the FnuDII isoschizomer BepI endonuclease isolated from this strain. The expression of BepI methylase in E. coli is dependent on the orientation of the cloned fragment suggesting that the gene is transcribed from a promoter on the plasmid vector. No BepI endonuclease could be detected in the clones producing BepI methylase. The nucleotide sequence of the BepI methylase gene has been determined, it predicts a protein of 403 amino acids (MR: 45,447). Analysis of the amino acid sequence deduced from the nucleotide sequence revealed similarities between the BepI methylase and other cytosine methylases. M. BepI methylates the external cytosine in its recognition sequence.

Amino Acid Sequence↗
PubMed1986

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator.

The dnaQ (mutD) gene product which encodes the epsilon-subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3'-5' exonucleolytic editing capacity. It is shown in this paper that more than 95% of all dnaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background. The introduction of a lexA (Ind-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect. Both, the mutational specificity observed and the partial lexA+ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.

Anticodon↗