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E H Postel

Publications and source records attributed to E H Postel.

At least 19 recordsLinked to original sources
PubMed1999

Cleavage of DNA by human NM23-H2/nucleoside diphosphate kinase involves formation of a covalent protein-DNA complex.

The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.

Antigens, Neoplasm↗
PubMed1998

NM23-NDP kinase.

NM23 belongs to a large family of structurally and functionally conserved proteins consisting of 4-6 identically folded subunits of approximately 16-20 kDa. These oligomeric proteins exhibit nucleoside diphosphate kinase (NDPK) activity that catalyzes nonsubstrate specific conversions of nucleoside diphosphates to nucleoside triphosphates. Many NM23 proteins bind DNA. In vivo, NM23-NDPKs regulate a diverse array of cellular events including growth and development. They are also implicated in the pathogenesis and metastasis of tumors. The mechanism whereby NM23 regulates gene expression is proposed to entail DNA-binding and subsequent alterations in promoter DNA structure. Accordingly, NM23 has the potential to become a useful reagent for gene manipulations.

Animals↗
PubMed1996

Mutational analysis of NM23-H2/NDP kinase identifies the structural domains critical to recognition of a c-myc regulatory element.

NM23-H2, a presumed regulator of tumor metastasis in humans, is a hexameric protein with both enzymatic (NDP kinase) and regulatory (transcriptional activation) activity. While the structure and catalytic mechanisms have been well characterized, the mode of DNA binding is not known. We examined this latter function in a site-directed mutational study and identified residues and domains essential for the recognition of a c-myc regulatory sequence. Three amino acids, Arg-34, Asn-69, and Lys-135, were found among 30 possibilities to be critical for DNA binding. Two of these, Asn-69 and Lys-135, are not conserved between NM23 variants differing in DNA-binding potential, suggesting that DNA recognition resides partly in nonconserved amino acids. All three DNA-binding defective mutant proteins are active enzymatically and appear to be stable hexamers, suggesting that they perform at the level of DNA recognition and that separate functional domains exist for enzyme catalysis and DNA binding. In the context of the known crystal structure of NM23-H2, the DNA-binding residues are located within distinct structural motifs in the monomer, which are exposed to the surface near the 2-fold axis of adjacent subunits in the hexamer. These findings are explained by a model in which NM23-H2 binds DNA with a combinatorial surface consisting of the "outer" face of the dimer. Chemical crosslinking data support a dimeric DNA-binding mode by NM23-H2.

Amino Acid Sequence↗
PubMed1995

PuF/NM23-H2/NDPK-B transactivates a human c-myc promoter-CAT gene via a functional nuclease hypersensitive element.

We have isolated the transacting factor PuF that, through its interaction with a nuclease hypersensitive element (NHE) located upstream of the c-myc gene, transactivates the human c-myc gene in vitro (Postel et al., 1989). PuF was recently identified as being encoded by the nonmetastatic 23-H2 (nm23-H2)/nucleoside diphosphate kinase-B (NDPK-B) gene (Postel et al., 1993). In addition to its ability to transactivate the c-myc gene in vitro, PuF/NDPK-B catalyzes the shuttling of gamma-phosphates between nucleoside triphosphates and diphosphates (Gilles et al., 1991; Postel and Ferrone, 1994) and has been postulated to suppress tumor metastasis (Stahl et al., 1991). Here we have extended our studies of PuF and c-myc transcription by testing whether PuF affects c-myc transcription using a transient transfection assay. A plasmid containing the human c-myc promoter-NHE region was cloned upstream of the bacterial chloramphenicol acetyltransferase (CAT) gene. When cotransfected with a PuF expression vector, CAT activity was elevated 3-4 fold relative to transfections containing the myc-CAT plasmid. In contrast, a myc-CAT reporter plasmid in which the NHE element was deleted showed no increase in CAT activity when cotransfected with the PuF expression vector. From these results we conclude that PuF transactivates the c-myc gene via the nuclease hypersensitive element.

Animals↗
PubMed1994

Nucleoside diphosphate kinase enzyme activity of NM23-H2/PuF is not required for its DNA binding and in vitro transcriptional functions.

nm23 genes encode proteins that participate in tumor metastasis regulation and in various fundamental cellular processes, although the mechanisms remain undefined. All Nm23 proteins contain nucleoside diphosphate kinase (NDPK) activity whose significance to these regulatory effects is not yet evident. The protein product of the human nm23-H2 gene functions in vitro both as a nucleoside diphosphate kinase enzyme (NDPK-B; Gilles, A.-M., Presecan, E., Vonica, A. and Lascu, I. (1991) J. Biol. Chem. 266, 8784-8789) and as a transcription factor (PuF; Postel, E. H., Berberich, S. J., Flint, S. J. and Ferrone, C. A. (1993) Science 261, 478-480). To understand the significance of these two biochemical activities to NM23-H2 function, we have investigated the relationship between the DNA binding and transcriptional activity of NM23-H2 and its NDPK function. Using site-directed mutagenesis of the cDNA encoding NM23-H2, we have created a mutant substituting for the amino acid histidine 118, the presumed site of phosphorylation in the formation of the phosphoenzyme intermediate, the nonphosphorylatable amino acid phenylalanine. The H118F mutant protein is shown to be catalytically inactive as measured both in a radioisotopic assay that detects formation of the phosphorylated enzyme intermediate and in a coupled enzyme assay that indicates nucleoside diphosphate formation. These results confirm that histidine 118 is the critical residue for NDPK-B activity. In addition, the H118F mutant protein lacking enzymatic activity displayed normal DNA binding affinity for the c-myc promoter in electrophoretic mobility shift assays, and retained full transcriptional activity using the c-myc gene in vitro. These results indicate a lack of correlation between nucleoside diphosphate kinase activity of nm23-H2 on the one hand, and its DNA binding and transcriptional activity on the other, suggesting that the nm23-H2 gene encodes a bifunctional protein molecule.

Base Sequence↗
PubMed1993

Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.

A human gene encoding the c-myc purine-binding transcription factor PuF was identified by screening of a cervical carcinoma cell complementary DNA library with a DNA fragment containing PuF binding sites. The 17-kilodalton bacterially produced PuF was shown to have biological activity and properties similar to that of human PuF. DNA sequence analysis of recombinant PuF revealed perfect identity with the human nm23-H2 nucleoside diphosphate kinase gene, a potential negative regulator of cancer metastasis. These results provide a link between nm23 and the c-myc oncogene and suggest that the nm23 protein can function in vitro in the transcriptional regulation of c-myc expression.

DNA-Binding Proteins↗
PubMed1992

Modulation of c-myc transcription by triple helix formation.

The human c-myc oncogene promoter was used as a model with which to study the mechanism of action of oligodeoxyribonucleotides targeted to a gene regulatory region. The nuclease-hypersensitive element, NHE, lying -115 bp from the P1 promoter of the human c-myc gene, is known to be required in cis for transcription of the gene from both P1 and P2 promoters (Fig. 1). Inhibition of c-myc transcription by an oligonucleotide designed to bind to NHE by triplex formation has been observed in a cell-free transcription assay. Using a reconstituted transcription system with the semipurified PuF transcription factor whose site of interaction resides within the NHE, it is shown here that the oligonucleotide inhibits PuF-mediated transcription. These findings, together with data presented elsewhere showing that: (1) PU1 binds to cloned DNA fragments to form a colinear triplex; (2) PU1 inhibits transcription in nuclear extracts; (3) triple helix formation inhibits the binding of PuF to its target NHE element in an in vitro binding competition assay (E. Postel, R. Durland, and M. Hogan, submitted); (4) triplex formation at the NHE target site can occur in living HeLa cells treated with the triplex-forming PU1 oligomer, and (5) c-myc mRNA synthesis in these treated cells is repressed, clearly support the proposed model in which the oligonucleotide targeted against the c-myc NHE promoter region binds to form a triplex, thereby blocking access to the regulatory protein PuF. This results in promoter-sensitive repression of transcriptional activation of the c-myc gene. The potential for manipulation of gene expression by oligonucleotides targeted to a DNA sequence of the c-myc oncogene promoter and other gene promoters is clear.

Base Sequence↗
PubMed1991

Evidence that a triplex-forming oligodeoxyribonucleotide binds to the c-myc promoter in HeLa cells, thereby reducing c-myc mRNA levels.

A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.

Base Sequence↗
PubMed1989

A nuclease-hypersensitive element of the human c-myc promoter interacts with a transcription initiation factor.

Transcription of the human c-myc oncogene is elaborately regulated, but the relevant molecular mechanisms are not yet understood. To begin to define elements and enzyme systems responsible for c-myc transcription in vitro, we partially purified a transcription factor essential for efficient and accurate in vitro initiation from the principal myc promoter, P2. DNA mobility shift assays located the factor binding domain at -142 to -115 with respect to the P1 promoter. This region contains pur/pyr sequences (predominantly purines in one strand), nuclease-hypersensitive sites (U. Siebenlist, L. Henninghausen, J. Battey, and P. Leder, Cell 37:381-391, 1984; C. Boles and M. Hogan, Biochemistry 26:367-376, 1987), and a triple-helix-forming element (M. Cooney, G. Czernuszewicz, E. Postel, S. Flint, and M. Hogan, Science 241:456-459, 1988). Methylation interference mapping established that the factor, termed PuF, directly contacts the repeated palindromic sequence GGGTGGG of the -142/-115 element. The interaction of PuF with this cis-acting element is necessary for P2 transcription in vitro, for (i) deletion of this 5' region from the myc promoter greatly reduced transcription efficiency and (ii) a synthetic duplex oligonucleotide corresponding to the -142/-115 sequence completely repressed c-myc transcription in the presence of the partially purified factor. These observations lend support to the hypothesis that pur/pyr sequences perform important biological roles in the regulation of c-myc gene expression, most likely by serving as transcription factor binding sites.

Base Composition↗
PubMed1988

Site-specific oligonucleotide binding represses transcription of the human c-myc gene in vitro.

A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

Electrophoresis↗
PubMed1985

Enhancement of genetic transformation frequencies of mammalian cell cultures by damage to the cell DNA.

Ultraviolet (UV)-light and 5-fluorodeoxyuridine (FUdR), two known DNA damaging agents, were found to enhance the frequency of stable plasmid transformations in several different animal cell lines. Combined treatment with the two agents was more effective than treatment with either agent alone. A correlation between the transformability of a cell line in the absence of treatment and its response to damaging treatment was also observed. Southern blot analysis of transformed clones indicated that the stimulation in transformation frequency was not due to an increased number of copies of the integrated plasmid in the transformed cells.

Animals↗
PubMed1972

Competence mutants. II. Physical and biological fate of donor transforming deoxyribonucleic acid.

Transformation-deficient (com(-)) mutants, which are able to bind donor transforming deoxyribonucleic acid (DNA) without yielding a significant number of transformants, were studied with regard to the fate of donor DNA. In no case was there any detectable degradation into acid-soluble radioactivity after donor DNA uptake. Physical experiments showed that some of these mutants are deficient in their ability to associate donor DNA with the recipient's chromosome (dad(-) mutants, for donor association defective), whereas others are able to form what appear to be normal donor-recipient complexes. In spite of physical evidence for integration, none of the dad(-) mutants contains biologically active recombinant DNA, suggesting that they might be deficient in the recombination process (dab(-) mutants, for donor association biologically defective). Donor biological activity is not replicated in any of the mutant strains, and in some cases there is a 10-fold reduction of donor transforming DNA within 60 min after DNA uptake.

Bacteriological Techniques↗
PubMed1972

Competence mutants. 3. Responses to radiations.

Class 3 com(-) mutants [normal in deoxyribonucleic acid (DNA) uptake but poor in ability to transform] were investigated with regard to ultraviolet (UV) and X-ray sensitivity of colony-forming ability and with regard to their ability to be transformed by UV- and X-ray-irradiated DNA. Three mutants, com(-)40, 60, and 78, were highly UV-sensitive in colony-forming ability. None of the mutants was more sensitive than wild type to UV-irradiated transforming DNA; in fact, six of the mutants showed considerably greater resistance. Two of the mutants (com(-)40 and 60) were slightly more sensitive to X ray in colony formation, whereas most of the mutants showed some degree of sensitivity to X-ray-irradiated transforming DNA. In addition, the physical fate of X-ray-irradiated transforming DNA has been examined, and in one case (com(-)48) there was a significant drop in sedimentation value of X-ray-irradiated donor DNA after uptake by recipient cells. The com(-) mutants analyzed have been classified on the basis of their UV and X-ray sensitivities, and, where appropriate, possible biochemical lesions have been implicated.

Bacteriological Techniques↗