We showed previously that bacterially expressed full-length human wild-type p53b(1-393) binds selectively to supercoiled (sc)DNA in sc/linear DNA competition experiments, a process we termed supercoil-selective (SCS) binding. Using p53 deletion mutants and pBluescript scDNA (lacking the p53 recognition sequence) at native superhelix density we demonstrate here that the p53 C-terminal domain (amino acids 347-382) and a p53 oligomeric state are important for SCS binding. Monomeric p53(361-393) protein (lacking the p53 tetramerization domain, amino acids 325-356) did not exhibit SCS binding while both dimeric mutant p53(319- 393)L344A and fusion protein GCN4-p53(347-393) were effective in SCS binding. Supershifting of p53(320-393)-scDNA complexes with monoclonal antibodies revealed that the amino acid region 375-378, constituting the epitope of the Bp53-10.1 antibody, plays a role in binding of the p53(320-393) protein to scDNA. Using electron microscopy we observed p53-scDNA nucleoprotein filaments produced by all the C-terminal proteins that displayed SCS binding in the gel electrophoresis experiments; no filaments formed with the monomeric p53(361- 393) protein. We propose a model according to which two DNA duplexes are compacted into p53-scDNA filaments and discuss a role for filament formation in recombination.
Target DNAs, including a 71-mer oligonucleotide, a PCR product and a plasmid DNA, all containing oligo(A) stretches, were hybridized at magnetic Dynabeads oligo(dT) 25 (DBT). The hybridization events were detected using a technique based on chemical modification of the target DNA with a complex of osmium tetroxide with 2,2'-bipyridine (Os, bipy) and voltammetric detection at carbon electrodes. DNA was modified with Os, bipy prior to capture at DBT, at the beads, or after release from the beads. In the latter case, DNA-Os, bipy was detected in the reaction mixture using adsorptive transfer stripping voltammetry involving extraction of unreacted Os, bipy from the electrode by organic solvents. Pre-labeling of the target plasmid DNA and the PCR product with Os, bipy significantly increased the yield of DNA captured at the beads. Tens of femtomoles of both short (the 71-mer oligonucleotide) and long (the 3-kilobase plasmid) target DNAs in a 20-microliter hybridization sample can be easily detected by means of these techniques. Various carbon electrode materials, including pyrolytic graphite (PGE), highly oriented pyrolytic graphite (HOPGE), carbon paste (CPE), glassy carbon and pencil graphite, were tested regarding their suitability for the detection of osmium-labeled DNA. Among them, PGE and HOPGE appeared usable in the measurements of both purified DNA-Os, bipy and its mixtures with unreacted Os, bipy while CPE was suitable for the detection purified osmium-labeled DNA.
The proteins streptavidin and avidin were electrochemically detected in solution by adsorptive transfer stripping square wave voltammetry (AdTS SWV) at a carbon paste electrode (CPE). AdTS SWV was used to quantify biotinylated oligonucleotides, DNA hybridizations, and avidin in extracts of transgenic avidin maize. The detection limits of denatured and native streptavidin were 6 pM and 120 nM, respectively. The results demonstrated that streptavidin/avidin AdTS SWV is a sensitive and specific method for quantifying DNA and proteins in biological samples such as foods and tissue extracts, including genetically modified crops (avidin maize) and other plants in neighboring fields.
We have compared the binding of human full-length p53 protein (p53; expressed in bacteria and insects) and its isolated core domain (p53CD, amino acids 94±312; expressed in bacteria) to negatively supercoiled (sc) DNA using gel electrophoresis and immunoblotting. Significant differences were observed; p53CD produced a relatively small and continuous retardation of scDNA, in contrast to the ladder of distinct bands formed by p53 in agarose gels. The ladder produced by full-length protein expressed in bacteria (p53b) was similar to that observed earlier with protein expressed in insect cells (p53i). Competition between scDNAs and their linearized (lin) forms showed a preference for scDNAs by both p53 and p53CD, but the ratios characterizing the distribution of the protein between sc and lin pBluescript DNAs were substantially higher for p53 (sc/lin . 60 in p53b) than for p53CD (sc/lin < 4).Strong binding of p53 to scDNA lacking the p53 consensus sequence may represent a new p53-binding mode, which we tentatively denote supercoil-selective (SCS) binding. This binding requires both the C-terminal domain and the core domain. Targets of this binding may include: (a) DNA segments defined both by the nucleotide sequence and local topology, and/or (b) strand crossings and/or bending. The binding preference of p53CD for scDNA may be due to the known nonspecific binding to internal single-stranded regions in scDNA (absent in relaxed DNA molecules) and/or to SCS binding albeit with reduced affinity due to the absence of contributions from other p53 domains.Keywords: tumor suppressor protein p53; comparison of full-length p53 and its isolated core domain; binding of p53 to supercoiled DNA; immunoblotting of p53.The p53 gene, frequently called the guardian of the genome, protects vulnerable cells from malignant transformation by regulating the responses of cell growth and death to genotoxic agents (reviewed in [1±5]). The functions of this gene are closely related to the specific binding of its product, the tumor suppressor protein p53, to the DNA consensus sequence (p53CON), consisting of two copies of the sequence 5 H -PuPuPuC(A / T)(T/A)GPyPyPy-3 H separated by 0±13 bp [6]. The p53 protein is a metalloprotein containing one zinc atom in the core domain [7,8]. The conformation of p53 is important for the biological activity of the protein and for its binding to p53CON. It has been suggested that the dual ability of p53 to function as a tumor suppressor and to promote cell proliferation may be due to switching between wild type and mutant conformations, respectively [9±11].Using agarose gel electrophoresis and scanning force microscopy (SFM) we have recently shown that the full-length wild-type human p53 protein binds strongly to negatively supercoiled DNA (scDNA) at native superhelix density [12]. The binding takes place both in the presence and absence of the consensus sequence (p53CON), producing several retarded bands on the gel. SFM images reveal partially or fully relaxed DNA molecules with bound p53 protein molecules....
Avidin and streptavidin were studied by phase-sensitive AC and cyclic voltammetry as well as by constant current chronopotentiometry at mercury (in alkaline media) and carbon electrodes (in acid medium). In contrast to the generally accepted belief that these proteins are electroinactive, we observed various electrochemical responses at these electrodes. Both proteins produced peaks due to oxidation of tyrosine and tryptophan residues at carbon electrodes and a catalytic peak H at a hanging mercury drop electrode. At the latter electrode avidin produced phasein AC voltammetric and cyclic voltammetric peaks close to À 0.6 V (peak S) which were assigned to Hg-S interactions, involving cystine/cysteine residues. In cobalt containing solution avidin produced a characteristic catalytic double wave requiring presence of cystine/cysteine residues in the protein molecule. Streptavidin, which does not contain these residues, yielded neither the catalytic double wave nor peak S. All the above avidin signals responded to biotin binding; peak S increased (up to 4 biotin molecules bound) while other avidin signals decreased as a result of biotin binding. A tentative scheme of interfacial behavior of avidin and avidin-biotin complex, depending on the electrode charge, was suggested.
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